Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation

ABSTRACT

The present invention provides tumor cell preparations for use as models of the EMT process for use in the identification of anti-cancer agents, wherein said tumor cell preparations comprise cells of the epithelial tumor cell line CFPAC-1, which are stimulated by receptor ligands to induce EMT, or which have been engineered to inducibly express a protein that stimulates EMT. The present invention also provides methods of identifying potential anti-cancer agents by using such tumor cell preparations to identify agents that inhibit EMT, stimulate MET, or inhibit the growth of mesenchymal-like cells. Such agents should be particularly useful when used in conjunction with other anti-cancer drugs such as EGFR and IGF-1R kinase inhibitors, which appear to be less effective at inhibiting tumor cells that have undergone an EMT.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/208,898, filed Feb. 27, 2009, which is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The present invention is directed to EMT cell models and methods fortheir use in the identification of new anti-cancer agents for treatingcancer patients, particularly in combination with other agents such asEGFR or IGF-1R kinase inhibitors that can be less effective atinhibiting tumor cells that have undergone an EMT. Cancer is a genericname for a wide range of cellular malignancies characterized byunregulated growth, lack of differentiation, and the ability to invadelocal tissues and metastasize. These neoplastic malignancies affect,with various degrees of prevalence, every tissue and organ in the body.

An anti-neoplastic drug would ideally kill cancer cells selectively,with a wide therapeutic index relative to its toxicity towardsnon-malignant cells. It would also retain its efficacy against malignantcells, even after prolonged exposure to the drug. Unfortunately, none ofthe current chemotherapies possess such an ideal profile. Instead, mostpossess very narrow therapeutic indexes. Furthermore, cancerous cellsexposed to slightly sub-lethal concentrations of a chemotherapeuticagent will very often develop resistance to such an agent, and quiteoften cross-resistance to several other antineoplastic agents as well.

A multitude of therapeutic agents have been developed over the past fewdecades for the treatment of various types of cancer. The most commonlyused types of anticancer agents include: DNA-alkylating agents (e.g.,cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, afolate antagonist, and 5-fluorouracil, a pyrimidine antagonist),microtubule disrupters (e.g., vincristine, vinblastine, paclitaxel), DNAintercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormonetherapy (e.g., tamoxifen, flutamide). More recently, gene targetedtherapies, such as protein-tyrosine kinase inhibitors have increasinglybeen used in cancer therapy (de Bono J. S. and Rowinsky, E. K. (2002)Trends in Mol. Medicine. 8:S19-S26; Dancey, J. and Sausville, E. A.(2003) Nature Rev. Drug Discovery 2:92-313). Such approaches, such asthe EGFR kinase inhibitor erlotinib, are generally associated withreduced toxicity compared with conventional cytotoxic agents. They aretherefore particularly appropriate for use in combination regimens. Inpancreatic cancer, phase III trials have shown that first-line erlotinibtreatment in combination with gemcitabine improves survival.

The epidermal growth factor receptor (EGFR) family comprises fourclosely related receptors (HER1/EGFR, HER2, HER3 and HER4) involved incellular responses such as differentiation and proliferation.Over-expression of the EGFR kinase, or its ligand TGF-alpha, isfrequently associated with many cancers, including breast, lung,colorectal, ovarian, renal cell, bladder, head and neck cancers,glioblastomas, and astrocytomas, and is believed to contribute to themalignant growth of these tumors. A specific deletion-mutation in theEGFR gene (EGFRvIII) has also been found to increase cellulartumorigenicity. Activation of EGFR stimulated signaling pathways promotemultiple processes that are potentially cancer-promoting, e.g.proliferation, angiogenesis, cell motility and invasion, decreasedapoptosis and induction of drug resistance. Increased HER1/EGFRexpression is frequently linked to advanced disease, metastases and poorprognosis. For example, in NSCLC and gastric cancer, increased HER1/EGFRexpression has been shown to correlate with a high metastatic rate, poortumor differentiation and increased tumor proliferation.

Mutations which activate the receptor's intrinsic protein tyrosinekinase activity and/or increase downstream signaling have been observedin NSCLC and glioblastoma. However the role of mutations as a principlemechanism in conferring sensitivity to EGF receptor inhibitors, forexample erlotinib (TARCEVA®) or gefitinib (IRESSA™), has beencontroversial. Recently, a mutant form of the full length EGF receptorhas been reported to predict responsiveness to the EGF receptor tyrosinekinase inhibitor gefitinib (Paez, J. G. et al. (2004) Science304:1497-1500; Lynch, T. J. et al. (2004) N. Engl. J. Med.350:2129-2139). Cell culture studies have shown that cell lines whichexpress the mutant form of the EGF receptor (i.e. H3255) were moresensitive to growth inhibition by the EGF receptor tyrosine kinaseinhibitor gefitinib, and that much higher concentrations of gefitinibwas required to inhibit the tumor cell lines expressing wild type EGFreceptor. These observations suggests that specific mutant forms of theEGF receptor may reflect a greater sensitivity to EGF receptorinhibitors, but do not identify a completely non-responsive phenotype.

Erlotinib (e.g. erlotinib HCl, also known as TARCEVA® or OSI-774) is anorally available inhibitor of EGFR kinase. In vitro, erlotinib hasdemonstrated substantial inhibitory activity against EGFR kinase in manyhuman tumor cell lines. In a phase III trial, erlotinib monotherapysignificantly prolonged survival, delayed disease progression anddelayed worsening of lung cancer-related symptoms in patients withadvanced, treatment-refractory NSCLC (Shepherd, F. et al. (2005) N.Engl. J. Med. 353(2):123-132). In November 2004 the U.S. Food and DrugAdministration (FDA) approved TARCEVA® for the treatment of patientswith locally advanced or metastatic non-small cell lung cancer (NSCLC)after failure of at least one prior chemotherapy regimen.

The development for use as anti-tumor agents of compounds that directlyinhibit the kinase activity of IGF-1R, as well as antibodies that reduceIGF-1R kinase activity by blocking IGF-1R activation or antisenseoligonucleotides that block IGF-1R expression, are also areas of intenseresearch effort (e.g. see Larsson, O. et al (2005) Brit. J. Cancer92:2097-2101; Ibrahim, Y. H. and Yee, D. (2005) Clin. Cancer Res.11:944s-950s; Mitsiades, C. S. et al. (2004) Cancer Cell 5:221-230;Camirand, A. et al. (2005) Breast Cancer Research 7:R570-R579 (DOI10.1186/bcr1028); Camirand, A. and Pollak, M. (2004) Brit. J. Cancer90:1825-1829; Garcia-Echeverria, C. et al. (2004) Cancer Cell5:231-239).

IGF-1R is a transmembrane RTK that binds primarily to IGF-1 but also toIGF-II and insulin with lower affinity. Binding of IGF-1 to its receptorresults in receptor oligomerization, activation of tyrosine kinase,intermolecular receptor autophosphorylation and phosphorylation ofcellular substrates (major substrates are IRS1 and Shc). Theligand-activated IGF-1R induces mitogenic activity in normal cells andplays an important role in abnormal growth. A major physiological roleof the IGF-1 system is the promotion of normal growth and regeneration.Overexpressed IGF-1R (type 1 insulin-like growth factor receptor) caninitiate mitogenesis and promote ligand-dependent neoplastictransformation. Furthermore, IGF-1R plays an important role in theestablishment and maintenance of the malignant phenotype. Unlike theepidermal growth factor (EGF) receptor, no mutant oncogenic forms of theIGF-1R have been identified. However, several oncogenes have beendemonstrated to affect IGF-1 and IGF-1R expression. The correlationbetween a reduction of IGF-1R expression and resistance totransformation has been seen. Exposure of cells to the mRNA antisense toIGF-1R RNA prevents soft agar growth of several human tumor cell lines.IGF-1R abrogates progression into apoptosis, both in vivo and in vitro.It has also been shown that a decrease in the level of IGF-1R belowwild-type levels causes apoptosis of tumor cells in vivo. The ability ofIGF-1R disruption to cause apoptosis appears to be diminished in normal,non-tumorigenic cells.

The IGF-1 pathway in human tumor development has an important role.IGF-1R overexpression is frequently found in various tumors (breast,colon, lung, sarcoma) and is often associated with an aggressivephenotype. High circulating IGF1 concentrations are strongly correlatedwith prostate, lung and breast cancer risk. Furthermore, IGF-1R isrequired for establishment and maintenance of the transformed phenotypein vitro and in vivo (Baserga R. Exp. Cell. Res., 1999, 253, 1-6). Thekinase activity of IGF-1R is essential for the transforming activity ofseveral oncogenes: EGFR, PDGFR, SV40 T antigen, activated Ras, Raf, andv-Src. The expression of IGF-1R in normal fibroblasts induces neoplasticphenotypes, which can then form tumors in vivo. IGF-1R expression playsan important role in anchorage-independent growth. IGF-1R has also beenshown to protect cells from chemotherapy-, radiation-, andcytokine-induced apoptosis. Conversely, inhibition of endogenous IGF-1Rby dominant negative IGF-1R, triple helix formation or antisenseexpression vector has been shown to repress transforming activity invitro and tumor growth in animal models.

During most cancer metastases, an important change occurs in a tumorcell known as the epithelial-mesenchymal transition (EMT) (Thiery, J. P.(2002) Nat. Rev. Cancer 2:442-454; Savagner, P. (2001) Bioessays23:912-923; Kang Y. and Massague, J. (2004) Cell 118:277-279;Julien-Grille, S., et al. Cancer Research 63:2172-2178; Bates, R. C. etal. (2003) Current Biology 13:1721-1727; Lu Z., et al. (2003) CancerCell. 4(6):499-515)). EMT does not occur in healthy cells except duringembryogenesis. Epithelial cells, which are bound together tightly andexhibit polarity, give rise to mesenchymal cells, which are heldtogether more loosely, exhibit a loss of polarity, and have the abilityto travel. These mesenchymal cells can spread into tissues surroundingthe original tumor, as well as separate from the tumor, invade blood andlymph vessels, and travel to new locations where they divide and formadditional tumors. Recent research has demonstrated that epithelialcells respond well to EGFR and IGF-1R kinase inhibitors, but that afteran EMT the resulting mesenchymal-like cells are much less sensitive tosuch inhibitors. (e.g. Thompson, S. et al. (2005) Cancer Res.65(20):9455-9462; U.S. Patent Application 60/997,514). Thus there is apressing need for anti-cancer agents that can prevent or reverse tumorcell EMT events (e.g. stimulate a mesenchymal to epithelial transition(MET)), or inhibit the growth of the mesenchymal-like tumor cellsresulting from EMT. Such agents should be particularly useful when usedin conjunction with other anti-cancer drugs such as EGFR and IGF-1Rkinase inhibitors.

As human cancers progress to a more invasive, metastatic state, multiplesignaling programs regulating cell survival and migration programs areobserved depending on cell and tissue contexts (Gupta, G. P., andMassague, J. (2006) Cell 127, 679-695). Recent data highlight thetransdifferentiation of epithelial cancer cells to a moremesenchymal-like state, a process resembling epithelial-mesenchymaltransition (EMT; (Oft, M., et al. (1996). Genes & development 10,2462-2477; Perl, A. K., et al. (1998). Nature 392, 190-193), tofacilitate cell invasion and metastasis (Brabletz, T. et al. (2005) NatRev Cancer 5, 744-749; Christofori, G. (2006) Nature 441, 444-450).Through EMT-like transitions mesenchymal-like tumor cells are thought togain migratory capacity at the expense of proliferative potential. Amesenchymal-epithelial transition (MET) has been postulated toregenerate a more proliferative state and allow macrometastasesresembling the primary tumor to form at distant sites (Thiery, J. P.(2002) Nat Rev Cancer 2, 442-454). EMT-like transitions in tumor cellsresult from transcriptional reprogramming over considerable periods oftime (weeks to months) via transcription factors harboring zinc finger,forkhead, bHLH and HMG-box domains (Mani, S. A. et al. (2007)Proceedings of the National Academy of Sciences of the United States ofAmerica 104, 10069-10074; Peinado, H. et al. (2007) Nat Rev Cancer 7,415-428). The loss of E-cadherin and transition to a moremesenchymal-like state likely serves a major role in the progression ofcancer (Matsumura, T. et al. (2001) Clin Cancer Res 7, 594-599;Yoshiura, K. et al. (1995). Proceedings of the National Academy ofSciences of the United States of America 92, 7416-7419) and theacquisition of a mesenchymal phenotype has been correlated with poorprognosis (Baumgart, E. et al. (2007) Clin Cancer Res 13, 1685-1694;Kokkinos, M. I. Et al. (2007) Cells, tissues, organs 185, 191-203;Willipinski-Stapelfeldt, B. et al. (2005) Clin Cancer Res 11,8006-8014.). Targeting tumor-derived and/or tumor-associated stromalcells provides a unique mechanism to block EMT-like transitions andinhibit the survival of invading cells.

The cellular changes associated with EMT-like transitions alter thedependence of carcinoma cells on EGF receptor signaling networks forsurvival. It has been observed that an EMT-like transition wasassociated with cellular insensitivity to the EGFR-TKI erlotinib(Thomson, S. et al. (2005) Cancer research 65, 9455-9462; Witta, S. E.,et al. (2006) Cancer research 66, 944-950; Yauch, R. L., et al. (2005)Clin Cancer Res 11, 8686-8698), in part from EGFR independent activationof either or both the PI3′ kinase or Mek-Erk pathways (Buck, E. et al.(2007). Molecular cancer therapeutics 6, 532-541). Similar datacorrelating EMT status to sensitivity to EGFR TKIs have been reported inpancreatic, CRC (Buck, E. et al. (2007) Molecular cancer therapeutics 6,532-541) bladder (Shrader, M. et al. (2007) Molecular cancertherapeutics 6, 277-285) and HNSCC (Frederick et al. (2007) Molecularcancer therapeutics 6, 1683-1691) cell lines, xenografts and in patients(Yauch, R. L., et al. (2005) Clin Cancer Res 11, 8686-8698). Themolecular determinants to alternative routes of activation of the PI3′kinase and Erk pathways, which can bypass cellular sensitivity to EGFreceptor inhibitors, have been actively investigated (Chakravarti, A. etal. (2002) Cancer research 62, 200-207; Engelman, J. A. et al. (2007)Science 316:1039-1043).

Inhibition of EMT-like transitions and mesenchymal-like cell survivalwould be predicted to reduce tumor metastasis and progression. Currentdata suggest patients with metastasis have heterogeneous tumors that cancontain cells with epithelial and mesenchymal-like phenotypes. Theobservation that tumors can acquire new signaling pathways, for examplePDGFR and FGFR autocrine signaling, suggest new therapeutic modalitiesto target specific tumor cell populations. These data suggest rationaldrug combinations that would not only cause growth inhibition orapoptosis of tumor cells directly, but would also impact mesenchymalcell populations promoting cancer recurrence (Moody, S. E. et al.(2005). Cancer cell 8, 197-209). Essential for the discovery anddevelopment of such drug combinations will be the availability of goodcellular and animal models where their efficacy can be readily assessed.The invention described herein provides such models.

SUMMARY OF THE INVENTION

The present invention provides a method of identifying an agent thatinhibits tumor cells from undergoing an epithelial to mesenchymaltransition, comprising contacting a sample of cells of the epithelialtumor cell line CFPAC-1 with a test agent to be screened, contacting thesample with a single protein ligand preparation that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells, determiningwhether the test agent inhibits the tumor cells in the sample fromundergoing an epithelial to mesenchymal transition, by comparing thelevel of a biomarker whose level is indicative of the EMT status of thesample tumor cells to the level of the same biomarker in an identicalsample of CFPAC-1 cells not contacted with the test agent, and thusdetermining whether the test agent is an agent that inhibits tumor cellsfrom undergoing an epithelial to mesenchymal transition. In oneembodiment, the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is selected fromOSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agentthat inhibits tumor cells that have undergone an epithelial tomesenchymal transition, comprising contacting a sample of cells of theepithelial tumor cell line CFPAC-1 with a single protein ligandpreparation to induce an epithelial-to-mesenchymal transition in theCFPAC-1 cells, contacting the sample of cells with a test agent to bescreened, determining whether the test agent inhibits mesenchymal-likeCFPAC-1 cell growth, and thus determining whether it is an agent thatinhibits the growth of tumor cells that have undergone an epithelial tomesenchymal transition. In one embodiment, the single protein ligandthat induces an epithelial-to-mesenchymal transition in CFPAC-1 cells isselected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33;PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agentthat stimulates mesenchymal-like tumor cells to undergo a mesenchymal toepithelial transition, comprising contacting a sample of cells of theepithelial tumor cell line CFPAC-1 with a single protein ligandpreparation to induce an epithelial-to-mesenchymal transition in theCFPAC-1 cells, contacting the sample of cells with a test agent to bescreened, determining whether the test agent stimulates themesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal toepithelial transition, by comparing the level of a biomarker whose levelis indicative of the EMT status of the sample tumor cells to the levelof the same biomarker in an identical sample of mesenchymal-like CFPAC-1cells not contacted with the test agent, and thus determining whetherthe test agent is an agent that stimulates mesenchymal-like tumor cellsto undergo a mesenchymal to epithelial transition. In one embodiment,the single protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF;PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a mesenchymal-like tumor cellpreparation for use in the identification of anti-cancer agents, whereinsaid tumor cell preparation is prepared by a process comprising:contacting a sample of cells of the epithelial tumor cell line CFPAC-1with a single protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, wherein thesingle protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF;PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a tumor cell preparation for use inthe identification of anti-cancer agents, wherein said tumor cellpreparation comprises: a sample of cells of the epithelial tumor cellline CFPAC-1, which have been engineered to inducibly express a proteinthat stimulates an epithelial to mesenchymal transition in CFPAC-1cells. The protein that is inducibly expressed and stimulates anepithelial to mesenchymal transition in CFPAC-1 cells may be Snail,Zeb-1, HGF or OSM. The present invention also provides methods ofidentifying potential anti-cancer agents by using such tumor cellpreparations to identify agents that inhibit EMT, stimulate MET, orinhibit the growth of mesenchymal-like cells.

The present invention also provides methods for preparing compositionscomprising agents identified by any of the methods described herein, tobe used in the treatment of tumors or tumor metastases.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1: Induction of EMT in CFPAC-1 cells by growth factors andcytokines. CFPAC-1 cells were treated with growth factors or cytokinesfor 7 days (see Materials and Methods), and protein biomarkers of EMT,E-cadherin and vimentin measured by immunoblotting of cell extracts.Factors included: SDF-1a (stromal derived factor-1), TRANCE (TNF-relatedactivation induced cytokine), FGF1 (fibroblast growth factor acidic),FGF2 (fibroblast growth factor basic), BMP4 (bone morphogenetic protein4), BMP7 (bone morphogenetic protein 7), IGF2 (insulin-like growthfactor 2), OSM (Oncostatin M), NRG1 (neuregulin-1), PDGFaa (plateletderived growth factor a), PDGFbb (platelet derive growth factor b), Ang2(angiopoeitin 2), amphiregulin, HMGB 1 (Human high-mobility group box 1protein), LIF (Leukemia inhibitory factor), GM-CSF (Granulocytemacrophage colony stimulating factor), M-CSF (Macrophage colonystimulating factor), PAR1 (Protease activated receptor 1 agonist), PAR2(Protease activated receptor 2 agonist), PAR4 (Protease activatedreceptor 4 agonist), HGF (Hepatocyte Growth Factor), IL-31(Interleukin-31), IL-33 (Interleukin-33), IL 1-a (Interleukin-1 alpha),CTGF (Connective tissue growth factor), WISP1 (Wnt-1 inducible signalingpathway protein), PTHrP (Polypeptide hormone-related protein), MCP1(monocyte chemotactic protein-1), and Fulvestrant at 1 μM.

FIG. 2: Titration of HGF and OSM treatment of CFPAC1 cells. Varyingconcentrations of HGF or OSM was used and the protein biomarkers wereanalysed by immunoblotting analysis of E-cadherin and vimentin inlysates of CFPAC-1 cells.

FIG. 3: CFPAC-1 morphology changes with HGF and OSM induction of EMT.CFPAC-1 cells were treated with HGF or OSM for 7 days and cellmorphology was determined by light microscopy. Control untreated CFPAC1cells formed colonies while HGF and OSM treated cells have were lesscompact with more elongated cell morphology.

FIG. 4: Confocal images of EMT induced changes in CFPAC-1 cells. CFPAC-1cells were treated with HGF, OSM or combination of HGF and OSM for 7days and stained for localization of E-cadherin (green) and vimentin(red). Absence of E-cadherin expression or mislocalization of E-cadherinto the cytoplasm and more pronounced staining of vimentin was observedwith growth factor or cytokine treatment as a result of EMT.

FIG. 5: Changes in migration and invasion of CFPAC-1 cells induced toundergo EMT. CFPAC-1 cells were treated with HGF, OSM or the combinationof HGF and OSM for 7 days. (A) CFPAC-1 cells were monitored for theircapacity to migrate in response to FBS with HGF (p value=0.04), OSM (pvalue <0.0001) and HGF+OSM (p value <0.0001). (B) CFPAC-1 cells weremonitored for their capacity to invade through collagen I in response toFBS (p value of <0.0001 for all treatments). (C)CFPAC-1 cells weremonitored for their capacity to invade through BME (basement membraneextract) in response to FBS (HGF, p value=0.03, OSM p value=0.002,HGF+OSM, p value=0.003).

FIG. 6: EMT induced in CFPAC-1 cells can be reversed resulting in amesenchymal to epithelial transition (MET). (A) CFPAC-1 cells weretreated with HGF, OSM or HGF+OSM for 14 days (D14). Ligand was removedand monitored for ability of cells to revert to a more epithelialmorphology. Cells were monitored on day 3 (D17), day 7 (D21) and day 12(D26) after ligand removal. Cells reverted to a more epithelialmorphology with complete reversion with 12 days of ligand withdrawal.(B) Immunoblotting analysis was performed with CFPAC cell lysatescorresponding to samples in panel A. E-cadherin is prominent beginningon day 3 of ligand withdrawal (labeled Day 17) and complete absence ofvimentin is observed after 12 days of ligand withdrawal (labeled Day26).

FIG. 7: Induction of EMT in CFPAC-1 with OSM can be inhibited with a JAKsmall molecule inhibitor. CFPAC-1 cells treated for 7 days with HGF orOSM in presence or absence of JAK small molecule inhibitor (0.25 μM) orMEK small molecule inhibitor (3 μM). JAK inhibitor blocked themorphology change when CFPAC-1 cells were treated with OSM (Top panel oflight microscopy). Protein biomarkers were analysed by immunoblottinganalysis of E-cadherin and vimentin.

FIG. 8: Immunofluorescence of E-cadherin and vimentin in CFPAC1 cells.Cells were treated for 7 days with the indicated ligands and stained forE-cadherin and vimentin as described. All treatments result indownregulation of E-cadherin, loss of cell-cell contacts andupregulation of vimentin.

FIG. 9: Impact of EMT on 3D growth. CFPAC1 cells were grown in Matrigelfor 14 days with or without HGF+OSM. Cells were then fixed and stainedfor E-cadherin and vimentin with TOPRO3 as a nuclear counterstain. EMTcorrelates with downregulation of E-cadherin expression, an increase invimentin expression and a decreased organization of colony architecture.

FIG. 10: EMT characteristics of CFPAC1 model in vivo. (A) WesternBlotting and PCR of E-cadherin and vimentin. CFPAC1 cells were implantedorthotopically in nude mice and allowed to grow for 2, 4 or 8 weeksbefore harvesting. Protein lysates were prepared from tumors andimmunoblotted for E-Cadherin and vimentin. The western blot shows 4individual tumors, and the PCR result is mean fold change in mRNAbetween 4 tumors from week 2 to week 8. (B) Immunohistochemistry ofE-cadherin and vimentin in CFPAC in a representative orthotopic 8 weektumor. Tumors sections were stained for E-cadherin and vimentin.

DETAILED DESCRIPTION OF THE INVENTION

The term “cancer” in an animal refers to the presence of cellspossessing characteristics typical of cancer-causing cells, such asuncontrolled proliferation, immortality, metastatic potential, rapidgrowth and proliferation rate, and certain characteristic morphologicalfeatures. Often, cancer cells will be in the form of a tumor, but suchcells may exist alone within an animal, or may circulate in the bloodstream as independent cells, such as leukemic cells.

“Cell growth”, as used herein, for example in the context of “tumor cellgrowth”, unless otherwise indicated, is used as commonly used inoncology, where the term is principally associated with growth in cellnumbers, which occurs by means of cell reproduction (i.e. proliferation)when the rate the latter is greater than the rate of cell death (e.g. byapoptosis or necrosis), to produce an increase in the size of apopulation of cells, although a small component of that growth may incertain circumstances be due also to an increase in cell size orcytoplasmic volume of individual cells. An agent that inhibits cellgrowth can thus do so by either inhibiting proliferation or stimulatingcell death, or both, such that the equilibrium between these twoopposing processes is altered.

“Tumor growth” or “tumor metastases growth”, as used herein, unlessotherwise indicated, is used as commonly used in oncology, where theterm is principally associated with an increased mass or volume of thetumor or tumor metastases, primarily as a result of tumor cell growth.

“Abnormal cell growth”, as used herein, unless otherwise indicated,refers to cell growth that is independent of normal regulatorymechanisms (e.g., loss of contact inhibition). This includes theabnormal growth of: (1) tumor cells (tumors) that proliferate byexpressing a mutated tyrosine kinase or over-expression of a receptortyrosine kinase; (2) benign and malignant cells of other proliferativediseases in which aberrant tyrosine kinase activation occurs; (4) anytumors that proliferate by receptor tyrosine kinases; (5) any tumorsthat proliferate by aberrant serine/threonine kinase activation; and (6)benign and malignant cells of other proliferative diseases in whichaberrant serine/threonine kinase activation occurs.

The term “treating” as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing,either partially or completely, the growth of tumors, tumor metastases,or other cancer-causing or neoplastic cells in a patient with cancer.The term “treatment” as used herein, unless otherwise indicated, refersto the act of treating.

The phrase “a method of treating” or its equivalent, when applied to,for example, cancer refers to a procedure or course of action that isdesigned to reduce or eliminate the number of cancer cells in an animal,or to alleviate the symptoms of a cancer. “A method of treating” canceror another proliferative disorder does not necessarily mean that thecancer cells or other disorder will, in fact, be eliminated, that thenumber of cells or disorder will, in fact, be reduced, or that thesymptoms of a cancer or other disorder will, in fact, be alleviated.Often, a method of treating cancer will be performed even with a lowlikelihood of success, but which, given the medical history andestimated survival expectancy of an animal, is nevertheless deemed anoverall beneficial course of action.

The term “therapeutically effective agent” means a composition that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician.

The term “therapeutically effective amount” or “effective amount” meansthe amount of the subject compound or combination that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician.

The present invention derives from research that provided methods fordetermining which tumors will respond most effectively to treatment withprotein-tyrosine kinase inhibitors (e.g. Thompson, S. et al. (2005)Cancer Res. 65(20):9455-9462; U.S. Patent Application 60/997,514) basedon whether the tumor cells have undergone an epithelial to mesenchymaltransition (“EMT”; Thiery, J. P. (2002) Nat. Rev. Cancer 2:442-454;Savagner, P. (2001) Bioessays 23:912-923; Kang Y. and Massague, J.(2004) Cell 118:277-279; Julien-Grille, S., et al. Cancer Research63:2172-2178; Bates, R. C. et al. (2003) Current Biology 13:1721-1727;Lu Z., et al. (2003) Cancer Cell. 4(6):499-515). This researchdemonstrated that epithelial cells respond well to EGFR and IGF-1Rkinase inhibitors, but that after an EMT the resulting mesenchymal-likecells are much less sensitive to such inhibitors. Biomarkers can be usedto determine whether tumor cells have undergone an EMT (Thomson, S. etal. (2005) Cancer Res. 65(20):9455-9462). As a result of such work itbecame apparent that new therapeutic approaches would be necessary tofind agents that were capable of inhibiting the genesis, growth and/orfunction of such mesenchymal-like cells, which are thought to be animportant element in the invasive and metastatic properties of tumors.

A considerable body of work is emerging that is beginning to delineatethe biochemical pathways involved in regulating tumor EMT events, and tocharacterize the resultant mesenchymal-like tumor cells. For example,experiments using specific siRNA inhibitors of the expression of variousprotein products produced by mesenchymal-like tumor cells havedemonstrated that reduced expression of the products of certain genescan specifically inhibit the growth of mesenchymal-like tumor cells.Thus pharmacological agents that also specifically inhibit theexpression of the protein products encoded by these genes, orspecifically inhibit the biological activity of the expressed proteins(e.g. phosphotransferase activity), such as specific antibodies toexpressed proteins that possess an extracellular domain, antisensemolecules, ribozymes, or small molecule enzyme inhibitors (e.g. proteinkinase inhibitors), are similarly expected to be agents that will alsospecifically inhibit the growth of mesenchymal-like tumor cells. Theanti-tumor effects of a combination of an EGFR or IGF-1R kinaseinhibitor with such an agent should be superior to the anti-tumoreffects of these kinase inhibitors by themselves, since such acombination should effectively inhibit both epithelial andmesenchymal-like tumor cells, and thus co-administration of such agentswith EGFR or IGF-1R kinase inhibitors should be effective for treatmentof patients with advanced cancers such as NSCL, pancreatic, colon orbreast cancers.

Given the identification of key targets for the discovery anddevelopment of agents that will inhibit the growth of mesenchymal-liketumor cells, or the EMT process, there is thus a pressing need formodels and methods to evaluate agents identified by in vitro screeningmethods (e.g. using protein kinase assays) to determine if they have thepredicted effect of inhibiting the growth and/or migration ofmesenchymal-like tumor cells, both at a cellular level, and in vivo. Thedata presented in the Examples herein demonstrates that the epithelialtumor cell line CFPAC-1 can be stimulated to undergo EMT by contactingit with one or more cell receptor ligands, or by inducing the expressionof one or more protein products capable of modulating the EMT process.Not all epithelial tumor cell lines can be stimulated to undergo EMT,and the optimal process for EMT induction is cell line dependent. Thus,CFPAC-1 tumor cells, treated in various ways to induce EMT, can be usedas models, both in vitro and vivo, in methods to identify agents thatcan inhibit mesenchymal-like tumor cells, prevent their formation, orreverse the EMT process. These methods are also useful in theidentification of agents for the treatment of fibrotic disordersresulting in part from EMT transitions, including but not limited torenal fibrosis, hepatic fibrosis, pulmonary fibrosis, and mesotheliomas.Thus any of the inventions described herein as being applicable to tumorcells, will also be applicable to other cell types involved in fibroticdiseases that undergo EMT. Similarly, any of the inventions describedherein as being useful for the identification of anti-cancer agents,will also be useful in the identification of anti-fibrotic agents fortreating diseases that involve fibrosis.

“CFPAC-1 cells” as used herein, refers to cells of the pancreaticepithelial cell line CFPAC-1 [a.k.a. CRL-1918™] available from theAmerican Tissue Culture Collection (ATCC) as ATCC® Number:CRL-1918™,derived from a pancreatic ductal adenocarcinoma (liver metastasis) froma patient with cystic fibrosis.

Accordingly, the present invention provides a method of identifying anagent that inhibits tumor cells from undergoing an epithelial tomesenchymal transition, comprising contacting a sample of cells of theepithelial tumor cell line CFPAC-1 with a test agent to be screened,contacting the sample with a single protein ligand preparation thatinduces an epithelial-to-mesenchymal transition in CFPAC-1 cells,determining whether the test agent inhibits the tumor cells in thesample from undergoing an epithelial to mesenchymal transition, bycomparing the level of a biomarker whose level is indicative of the EMTstatus of the sample tumor cells to the level of the same biomarker inan identical sample of CFPAC-1 cells not contacted with the test agent,and thus determining whether the test agent is an agent that inhibitstumor cells from undergoing an epithelial to mesenchymal transition. Inone embodiment, the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is selected fromOSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; and BMP4.

A “single protein ligand preparation” as used herein, means apreparation comprising a protein ligand for a cell receptor, whichligand is capable, by itself, of substantially inducing EMT in CFPAC-1cells, as assessed for example by a significant decrease in expressionof the epithelial biomarker E-cadherin, and/or a significant increase inexpression of the mesenchymal biomarker vimentin. A single proteinligand preparation may contain additional compounds or proteins, e.g.cell nutrients, other growth factors, agents that stabilize the proteinligands, etc. For example, a single protein ligand preparation may besupplemented with one or more alternative single ligands that alsoinduce EMT, or a ligand that does not normally induce EMT on its own,which may result in a slightly more complete EMT. By contrast withCFPAC-1 cells, induction of EMT in other cells can sometimes require a“dual protein ligand preparation”, i.e. a preparation comprising twoprotein ligands for different cell receptors, which ligands are capableof inducing EMT in cells, and where both ligands are required for EMTinduction (i.e. either ligand by itself does not substantially induceEMT) (e.g. EMT induction in H358 cells by HGF and OSM, see U.S.provisional application 61/068,612).

In any of the methods or cell preparations of the invention describedherein, the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells may be selectedfrom any of the protein ligands that bind to and activate the receptorsfor OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4agonist AYPGKF-NH₂; CTGF; and BMP4, either presently known, or yet to bediscovered or synthesized. For example, the single protein ligand thatinduces an epithelial-to-mesenchymal transition in CFPAC-1 cells may beselected from OSM (Oncostatin M; NCIB GeneID: 5008); HGF (hepatocytegrowth factor; NCIB GeneID: 3082); BMP7 (bone morphogenetic protein 7;NCIB GeneID: 655); IGF2 (insulin-like growth factor 2 (a.k.a.somatomedin A); NCIB GeneID: 3481); LIF (Leukemia inhibitory factor;NCIB GeneID: 3976); PAR2 agonists, e.g. SLIGKV-NH₂; IL-33(Interleukin-33; NCIB GeneID: 90865); PAR4 agonists, e.g. AYPGKF-NH₂;CTGF (Connective tissue growth factor; NCIB GeneID: 1490); and BMP4(bone morphogenetic protein 4; NCIB GeneID: 652). In a preferredembodiment the single protein ligand is HGF or OSM; either optionallysupplemented with one of BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂;IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4. In an alternativeembodiment, the single protein ligand is a ligand selected from OSM;HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; or BMP4; optionally supplemented with one of OSM; HGF;BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; or BMP4. The human versions of the above ligandproteins are preferred, but where an alternative animal version exists(e.g. from mouse, rat, rabbit, dog, monkey, pig, etc) that also hasactivity in stimulating the human receptor on CFPAC-1 cells, and inducesEMT, this may also be used.

In any of the methods or cell preparations of the invention describedherein, when a more complete EMT is sought, as judged by E-cadherin orvimentin expression, the preferred single protein ligands to induce anepithelial-to-mesenchymal transition in CFPAC-1 cells may comprise aligand that binds to and activates the oncostatin-M receptor (e.g. OSM)or a ligand that binds to and activates the HGF receptor (a.k.a. Metreceptor tyrosine kinase) (e.g. HGF). In an alternative embodiment, thesingle protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells may comprise a protein ligand that binds toa receptor that activates the signal transduction pathways activated bythe binding of oncostatin M to its receptor (e.g. a ligand that binds toand activates the oncostatin-M receptor (e.g. oncostatin M), or aprotein ligand that binds to another receptor that activates the samesignal transduction pathways as are activated by binding of oncostatin Mto its receptor (i.e. JAK-STAT pathways)); or a ligand that binds to acell tyrosine kinase receptor and activates the same signal transductionpathways that are activated by the binding of HGF to its receptor (i.e.the PI3K and MAPK pathways; as activated by binding of HGF to Metreceptor tyrosine kinase).

Receptor tyrosine kinases that activate the PI3K and MAPK pathwaysinclude for example, IGF1-R, FGFR1, FGFR2, FGFR3, FGFR4, heterodimers ofFGF receptors 1-4, RON, EGFR, HER-4, heterodimers of HER receptors 1-4,VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), VEGFR-3 (Flt-4), and PDGFR (α andβ receptor homo- and herero-dimers). Thus, examples of additionalligands that may induce an epithelial-to-mesenchymal transition inCFPAC-1 cells include IGF-1, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7,FGF8, FGF8, FGF10, macrophage-stimulating protein (MSP; RON receptorligand), transforming growth factor-α (TGF-α), heparin-binding EGF-likegrowth factor (HB-EGF), amphiregulin (AREG), betacellulin (BTC),epiregulin (EREG), epigen (EPGN), neuregulins (NRG-1 (Heregulin), NRG-2,NRG-3, NRG-4), VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PDGF-AA, PDGF-AB,PDGF-BB, PDGF-CC, and PDGF-DD, where sufficient receptor levels arepresent. The human versions of the above ligand proteins are preferred,but where an alternative animal version exists (e.g. from mouse, rat,rabbit, dog, monkey, pig, etc) that also has activity in stimulating thehuman receptor on CFPAC-1 cells, and induces EMT, this may also be used.

The induction of EMT in CFPAC-1 cells by the specific ligands disclosedherein allows targeting of specific pathways that induce EMT, and thusfor identification of anticancer agents that may have different modes ofaction, and may thus act together in a synergistic manner.

The NCBI GeneID numbers listed herein are unique identifiers of the genefrom the NCBI Entrez Gene database record (National Center forBiotechnology Information (NCBI), U.S. National Library of Medicine,8600 Rockville Pike, Building 38A, Bethesda, Md. 20894; Internet addresshttp://www.ncbi.nlm.nih.gov/). They are used herein to unambiguouslyidentify gene products that are referred to elsewhere in the applicationby names and/or acronyms. Proteins expressed by genes thus identifiedrepresent proteins that may be used in the methods of this invention,and the sequences of these proteins, including different isoforms, asdisclosed in NCBI database (e.g. GENBANK®) records are hereinincorporated by reference.

The sample of cells of the epithelial tumor cell line CFPAC-1 in any ofthe methods or preparations of this invention can be for example cellsin monolayer culture (e.g. cells in a tissue culture plate or dish, e.g.a 96-well plate); cells in three-dimensional culture, such as forexample Matrigel™ 3D culture, spheroid cultures, or soft agar culture(Kim, J. B., (2005) Seminars in Cancer Biology 15:365-377; Sutherland,R. M., (1988) Science 240:177-184; Hamilton, G. (1998) Cancer Letters,131:29-34; or cells in vivo, e.g. a tumor xenograft. In methodsdescribed herein that require “an identical sample of cells”, thisrefers to a sample of cells with essentially the same number of cells,growing under the same conditions. For example, an identical tissueculture dish with approximately the same number of cells, or a tumorxenograft of the same or similar size.

Many biomarkers are known whose level of expression or activity isindicative of the EMT status of tumor cells (e.g. see US PatentApplication Publication 2007/0212738; U.S. Patent Application60/923,463; U.S. Patent Application 60/997,514). Such markers tend to beclassified as epithelial or mesenchymal, due to their characteristicassociation with the particular stage of EMT. Characteristic biomarkerscan be, for example, proteins, encoding mRNAs, activity of a genepromoter, level of a transcriptional repressor, or promoter methylation.In any of the methods described herein the biomarker whose expressionlevel is indicative of the EMT status of the sample tumor cells can bean epithelial cell biomarker. Epithelial cell biomarkers include forexample E-cadherin, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3.Additional epithelial cell biomarkers include Brk, γ-catenin,α1-catenin, α2-catenin, α3-catenin, connexin 31, plakophilin 3,stratifin 1, laminin alpha-5, and ST14. In any of the methods describedherein the biomarker whose expression level is indicative of the EMTstatus of the sample tumor cells can also be a mesenchymal cellbiomarker. Mesenchymal cell biomarkers include for example is vimentin,fibronectin, N-cadherin, zeb 1, twist, FOXC2 or snail. Additionalmesenchymal cell biomarkers include, fibrillin-1, fibrillin-2, collagenalpha2(IV), collagen alpha2(V), LOXL1, nidogen, C11orf9, tenascin,tubulin alpha-3, and epimorphin. Additionally any other epithelial ormesenchymal cell biomarkers known in the art, described herein, or yetto be described, may be used in the methods of the invention describedherein. In any of the methods described herein, multiple biomarker leveldeterminations can also be used to assess EMT status, potentiallyproviding a more reliable assessment. For example, an epithelial and amesenchymal biomarker level may be assessed, the reciprocal changes ineach providing internal confirmation that EMT has occurred (e.g.suitable biomarker pairs include for example, E-cadherin/vimentin). Inan alternative embodiment, the epithelial biomarker comprises one ormore keratins selected from the epithelial keratins 1-28 and 71-80, andthe mesenchymal biomarker is vimentin, wherein co-expression ofepithelial and mesenchymal biomarkers at similar levels is indicative ofa mesenchymal-like tumor cell (see U.S. Patent Application 60/923,463).When used in any of the methods of the invention described herein,epithelial keratins 1-28 and 71-80 includes all the keratins listed inTable 2 herein. In one embodiment of the latter method the epithelialkeratin(s) are assessed using a method that will detect all or themajority (i.e. 50% or more) of the keratin biomarkers expressed by thetumor cell (e.g. by using a multi- or pan-specific antibody). In anotherembodiment of above methods where epithelial keratin biomarker levelsare determined, the biomarker comprises keratin 8 and/or keratin 18.

The term “co-expression of epithelial and mesenchymal biomarkers atsimilar levels” as used herein in the context of determiningco-expression of epithelial keratins and the mesenchymal biomarkervimentin means that the ratio of mesenchymal to epithelial biomarkerlevels is in the range of about 10:1 to about 1:10 (assuming that eachbiomarker is assayed under comparable conditions, e.g. using antibodiesof identical affinity, nucleic acid probes of identical length,identical detection methods, etc.).

In an alternative embodiment of any of the methods described herein thatinclude a step of determining the level of a biomarker whose level isindicative of the EMT status of the sample tumor cells, the biomarkercan be the activity of a gene promoter that is altered when the tumorcells undergo EMT. Such promoter activity is readily assessed byincorporating a promoter-reporter construct into the tumor cells andmeasuring reporter activity. In one embodiment, the activity of anepithelial biomarker gene promoter is assessed by inclusion of anepithelial biomarker gene promoter-reporter gene construct into theCFPAC-1 cells such that said promoter reporter activity can be monitoredby reporter gene expression level or activity. For example, theepithelial biomarker gene promoter-reporter gene construct may be anE-cadherin promoter-firefly luciferase construct. In an alternativeembodiment, the activity of a mesenchymal biomarker gene promoter isassessed by inclusion of a mesenchymal biomarker gene promoter-reportergene construct into the CFPAC-1 cells such that said promoter reporteractivity can be monitored by reporter gene expression level or activity.For example, the mesenchymal biomarker gene promoter-reporter geneconstruct may be an vimentin promoter-firefly luciferase construct. Thepromoter-reporter gene construct may be permanently incorporated intothe CFPAC-1 cells as a stable engineered cell line, or may betransiently expressed, using any of the standard techniques fortransferring nucleic acid constructs into cells (e.g. transfection,electroporation). Multiple promoter-reporter gene constructs may also beemployed in order to monitor several biomarkers simultaneously, e.g. anE-cadherin promoter-firefly luciferase construct and a vimentinpromoter-renilla luciferase construct, in order to, for example, monitorsimultaneous repression of the E-cadherin gene and induction of thevimentin gene as tumor cells undergo EMT.

The present invention also provides a method of identifying an agentthat inhibits tumor cells that have undergone an epithelial tomesenchymal transition, comprising contacting a sample of cells of theepithelial tumor cell line CFPAC-1 with a single protein ligandpreparation to induce an epithelial-to-mesenchymal transition in theCFPAC-1 cells, contacting the sample of cells with a test agent to bescreened, determining whether the test agent inhibits mesenchymal-likeCFPAC-1 cell growth, and thus determining whether it is an agent thatinhibits the growth of tumor cells that have undergone an epithelial tomesenchymal transition. In one embodiment, the single protein ligandthat induces an epithelial-to-mesenchymal transition in CFPAC-1 cells isselected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33;PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4. An alternative embodiment ofthis method comprises, after the step of determining whether the testagent inhibits the growth of tumor cells that have undergone anepithelial to mesenchymal transition, the additional steps ofdetermining whether an agent that inhibits mesenchymal-like CFPAC-1tumor cell growth, also inhibits epithelial CFPAC-1 tumor cell growth,and thus determining whether it is an agent that specifically inhibitsthe growth of tumor cells that have undergone an epithelial tomesenchymal transition. In an embodiment of the above methods, an agentthat inhibits the growth of tumor cells that have undergone anepithelial to mesenchymal transition is determined to do so bystimulating apoptosis of said tumor cells. In another embodiment of theabove methods, an agent that inhibits the growth of tumor cells thathave undergone an epithelial to mesenchymal transition is determined todo so by inhibiting proliferation of said tumor cells.

The present invention also provides a method of identifying an agentthat stimulates mesenchymal-like tumor cells to undergo a mesenchymal toepithelial transition, comprising contacting a sample of cells of theepithelial tumor cell line CFPAC-1 with a single protein ligandpreparation to induce an epithelial-to-mesenchymal transition in theCFPAC-1 cells, contacting the sample of cells with a test agent to bescreened, determining whether the test agent stimulates themesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal toepithelial transition, by comparing the level of a biomarker whose levelis indicative of the EMT status of the sample tumor cells to the levelof the same biomarker in an identical sample of mesenchymal-like CFPAC-1cells not contacted with the test agent, and thus determining whetherthe test agent is an agent that stimulates mesenchymal-like tumor cellsto undergo a mesenchymal to epithelial transition. In one embodiment,the single protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF;PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of preparing a compositioncomprising a chemical compound which inhibits the growth of tumor cellsthat have undergone an epithelial to mesenchymal transition, whichcomprises contacting a sample of cells of the epithelial tumor cell lineCFPAC-1 with a test agent to be screened, contacting the sample with asingle protein ligand preparation that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells, determiningwhether the test agent inhibits the tumor cells in the sample fromundergoing an epithelial to mesenchymal transition, by comparing thelevel of a biomarker whose level is indicative of the EMT status of thesample tumor cells to the level of the same biomarker in an identicalsample of CFPAC-1 cells not contacted with the test agent, and thusdetermining whether the test agent is an agent that inhibits tumor cellsfrom undergoing an epithelial to mesenchymal transition, and admixingthe test agent so identified with a carrier, thereby preparing saidcomposition.

The present invention also provides a method of preparing a compositioncomprising a chemical compound which inhibits the growth of tumor cellsthat have undergone an epithelial to mesenchymal transition, whichcomprises contacting a sample of cells of the epithelial tumor cell lineCFPAC-1 with a single protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, contactingthe sample of cells with a test agent to be screened, determiningwhether the test agent inhibits mesenchymal-like CFPAC-1 cell growth,and thus determining whether it is an agent that inhibits the growth oftumor cells that have undergone an epithelial to mesenchymal transition,and admixing the test agent so identified with a carrier, therebypreparing said composition.

The present invention also provides a method of preparing a compositioncomprising a chemical compound which inhibits the growth of tumor cellsthat have undergone an epithelial to mesenchymal transition, whichcomprises contacting a sample of cells of the epithelial tumor cell lineCFPAC-1 with a single protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, contactingthe sample of cells with a test agent to be screened, determiningwhether the test agent stimulates the mesenchymal-like CFPAC-1 cells inthe sample to undergo a mesenchymal to epithelial transition, bycomparing the level of a biomarker whose level is indicative of the EMTstatus of the sample tumor cells to the level of the same biomarker inan identical sample of mesenchymal-like CFPAC-1 cells not contacted withthe test agent, and thus determining whether the test agent is an agentthat stimulates mesenchymal-like tumor cells to undergo a mesenchymal toepithelial transition, and admixing the test agent so identified with acarrier, thereby preparing said composition.

The present invention also provides a mesenchymal-like tumor cellpreparation for use in the identification of anti-cancer agents, whereinsaid tumor cell preparation is prepared by a process comprising:contacting a sample of cells of the epithelial tumor cell line CFPAC-1with a single protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, wherein thesingle protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF;PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

For any of the methods described herein, a test agent can be anychemical compound, including small molecules (<approx. 5000 Daltonsmolecular weight) and macromolecules (e.g. a polypeptide or protein,nucleic acid, glycoprotein, complex carbohydrate, synthetic or naturalpolymer etc.). Thus, a test agent may be selected from, for example,combinatorial libraries, defined chemical entities, peptide and peptidemimetics, oligonucleotides and natural product libraries, aptamers, andother entities such as display (e.g. phage display libraries) andantibody products.

The present invention also provides a method of identifying an agentthat inhibits tumor cells from undergoing an epithelial to mesenchymaltransition, comprising: contacting a sample of cells of the epithelialtumor cell line CFPAC-1 with a test agent to be screened, wherein thecells are implanted orthotopically into the pancreas of animmune-deficient animal, determining whether the test agent inhibits thetumor cells in the sample from undergoing an epithelial to mesenchymaltransition, by comparing the level of a biomarker whose level isindicative of the EMT status of the sample tumor cells to the level ofthe same biomarker in an identical sample of CFPAC-1 cells not contactedwith the test agent, and thus determining whether the test agent is anagent that inhibits tumor cells from undergoing an epithelial tomesenchymal transition. In one embodiment of this method theimmune-deficient animal is a nude mouse. In another embodiment, thebiomarker whose level is indicative of the EMT status of the sampletumor cells is an epithelial cell biomarker. The epithelial cellbiomarker may be, for example, E-cadherin, CDH1 promoter activity,cytokeratin 8, cytokeratin 18, P-cadherin, or erbB3. In anotherembodiment, the biomarker whose level is indicative of the EMT status ofthe sample tumor cells is a mesenchymal cell biomarker. The mesenchymalcell biomarker may be, for example, vimentin, fibronectin, N-cadherin,CDH1 methylation, zeb1, twist, FOXC2 or snail.

The present invention also provides an animal model for use in theidentification of anti-cancer agents, which comprises a sample of cellsof the epithelial tumor cell line CFPAC-1 which have been implantedorthotopically into the pancreas of an immuno-deficient animal. In oneembodiment, the immune-deficient animal is a nude mouse (also known as aFoxn1nu mouse).

The present invention also provides a method of identifying an agentthat inhibits tumor cells from undergoing an epithelial to mesenchymaltransition, comprising contacting a sample of cells of the epithelialtumor cell line CFPAC-1, which have been engineered to inducibly expressa protein that stimulates an epithelial to mesenchymal transition inCFPAC-1 cells, with a test agent to be screened, contacting the samplewith a compound that induces the expression of said protein thatstimulates an epithelial to mesenchymal transition in the engineeredCFPAC-1 cells, determining whether the test agent inhibits the tumorcells in the sample from undergoing an epithelial to mesenchymaltransition, by comparing the level of a biomarker whose level isindicative of the EMT status of the sample tumor cells to the level ofthe same biomarker in an identical sample of engineered CFPAC-1 cellsnot contacted with the test agent, and thus determining whether the testagent is an agent that inhibits tumor cells from undergoing anepithelial to mesenchymal transition.

“Inducibly express”, as used herein, when referring for example to cellswhich have been engineered to “inducibly express” a protein, means thatthe protein expression is only turned on by the presence (or absence) ofan inducing agent that controls transcription of the gene encoding theprotein, which will preferably be incorporated into the cells by stabletransformation with a construct containing the gene for the encodingprotein under the control of a promoter that is responsive to theinducing agent (i.e. the gene encoding the protein is operably linked toa nucleotide sequence regulating the gene expression, which nucleotidesequence comprises a promoter sequence whose activity can be controlledby the presence of an inducing agent). One example of such an induciblepromoter is a tetracycline (tet)-responsive promoter (e.g. a Tet-onsystem; e.g. see Gossen, M. et al. (1995) Science 268:1766-1769). Suchinducible gene expression systems for controlling the expression levelsof specific genes of interest are well known in the art (e.g. see Blau,H. M. and Rossi, F. M. V. (1999) Proc. Natl. Acad. Sci. USA 96:797-799;Yamamoto, A. et al. (2001) Neurobiology of Disease 8:923-932; Clackson,T. (2000) Gene Therapy 7:120-125).

For any of the methods or cell preparations described herein involvingthe epithelial tumor cell line CFPAC-1 which has been engineered toinducibly express a protein that stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells, in one embodiment the cells also comprise apromoter-reporter gene construct that can be similarly induciblyexpressed, such that said promoter reporter activity can be monitored byreporter gene expression level or activity, and thus be used to readilyassess whether induction of the protein that stimulates an epithelial tomesenchymal transition has been successful. In one example of such anembodiment, the reporter gene is the gene for firefly or Renillaluciferase. Assessment of reporter levels can for example be used forreadily monitoring the extent of EMT induction of CFPAC-1 cells, and thelocation of cells that have undergone EMT. Thus, for example, cells invivo that have migrated from a primary tumor, or metastasized, can bereadily tracked by monitoring such a reporter gene. Thus, the presentinvention provides a method of identifying an agent that inhibits tumorcells from undergoing an epithelial to mesenchymal transition (andmetastasis), comprising contacting in vivo a sample of cells of theepithelial tumor cell line CFPAC-1, which have been engineered toinducibly express both a protein that stimulates an epithelial tomesenchymal transition in CFPAC-1 cells, and a reporter gene product(e.g. firefly luciferase), and which have been allowed to form a tumorxenograft in an immunocompromised animal, with a test agent to bescreened, contacting the sample with a compound that induces theexpression of said protein that stimulates an epithelial to mesenchymaltransition in the engineered CFPAC-1 cells and the reporter geneproduct, determining whether the test agent inhibits the tumor cells inthe sample from undergoing an epithelial to mesenchymal transition (andmetastasis), by comparing the extent of migration of the sample tumorcells away from a primary tumor (e.g. by imaging analysis of thereporter gene product) to the extent of migration of an identical sampleof engineered CFPAC-1 cells not contacted with the test agent, and thusdetermining whether the test agent is an agent that inhibits tumor cellsfrom undergoing an epithelial to mesenchymal transition (andmetastasis). Cells derived form an EMT-transition in vivo can also bedetected by surgical isolation and immunohistochemistry or in situhybridization using for example biomarkers as described herein, or in USPatent Application Publication 2007/0212738, U.S. Patent Application60/923,463, U.S. Patent Application 61/068,612, or U.S. PatentApplication 60/997,514).

In one embodiment of the above methods the protein that is induciblyexpressed and stimulates an epithelial to mesenchymal transition inCFPAC-1 cells is Snail. In another embodiment of this method the proteinthat is inducibly expressed and stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells is Zeb-1. In another embodiment of thismethod the protein that is inducibly expressed and stimulates anepithelial to mesenchymal transition in CFPAC-1 cells is OSM; HGF; BMP7;IGF2; LIF; a PAR2 agonist; IL-33; a PAR4 agonist; CTGF; or BMP4. In analternative embodiment, the protein that is inducibly expressed andstimulates an epithelial to mesenchymal transition in CFPAC-1 cells maybe co-expressed with one or more other proteins that enhance the EMT.Additional EMT causing genes that encode proteins that may be induciblyexpressed in engineered CFPAC-1 cells to promote EMT include, but arenot limited to, constitutively active cMET receptor; and activated Srckinase (e.g. v-Src or Src Y530F mutants). Similarly, in any of the othermethods or cell preparations described herein involving a protein thatis inducibly expressed and stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells, the protein can be any of the examplesdescribed above.

In one embodiment of these methods, a Tet-regulated promoter is used toinducibly express the protein that stimulates an epithelial tomesenchymal transition in CFPAC-1 cells, e.g. a Tet-on system. In oneembodiment of this method, the compound that induces the expression ofthe protein that stimulates an epithelial to mesenchymal transition inthe engineered CFPAC-1 cells is doxycycline. Other inducers that may beused include, but are not limited to, tetracycline andanhydrotetracycline. Similarly, in any of the other methods or cellpreparations described herein involving a protein that is induciblyexpressed and stimulates an epithelial to mesenchymal transition inCFPAC-1 cells, the promoter and inducer compound used to induciblyexpress the protein can be any of the examples described above.Induction systems include but are not limited to tetracycline regulatedplasmids, e.g. Tet-on and Tet-off systems?

In the above methods the biomarker whose level is indicative of the EMTstatus of the sample tumor cells is for example an epithelial cellbiomarker, e.g. E-cadherin, cytoketatin 8, cytokeratin 18, P-cadherin orerbB3, or the activity of an epithelial biomarker gene promoter. In oneembodiment, the activity of an epithelial biomarker gene promoter may beassessed by inclusion of a human epithelial biomarker genepromoter-reporter gene construct in the engineered CFPAC-1 cells suchthat said promoter reporter activity can be monitored by reporter geneexpression level or activity. In one embodiment, the epithelialbiomarker gene promoter-reporter gene construct is a human E-cadherinpromoter-firefly luciferase construct. Additional examples of epithelialpromoters that may be used include the promoters of the following genes:human ELF3 (i.e. E74-like factor 3 (ets domain transcription factor,epithelial-specific), GeneID: 1999). In an alternative embodiment, RNAtranscript splicing mechanisms that are unique to human epithelial cells(Savagner, P. et al. (1994) Mol Biol Cell. 5(8):851-862; Oltean, S. etal. (2006) Proc Natl Acad Sci USA. 103(38):14116-14121; Ghigna, C. etal. (2005) Mol. Cell. 20(6):881-890; Bonano, V. I. et al. (2007) Nat.Protoc. 2(9):2166-2181), and do not operate after EMT, can be utilizedas markers of EMT status. For example, by including the sequencesnecessary for such epithelial-specific splicing into apromoter-luciferase construct incorporated into CFPAC-1 cells (e.g. aCMV promoter-firefly luciferase construct) such that active luciferasewill only be expressed in the epithelial state, induction of EMT canreadily be monitored by a decrease in luciferase expression or activity.In an alternative embodiment, miRNAs that are expressed specifically inhuman epithelial cells (e.g. see Hurteau, G. J. et al. (2007) CancerResearch 67:7972-7976; Christoffersen, N. R. et al. (2007) RNA13:1172-1178; Shell, S. et al (2007) Proc Natl. Acad. Sci.104(27):11400-11405), that degrade or diminish translation oftranscripts containing complementary nucleic acid sequences, can beutilized as markers of EMT status. For example, by including sequencescomplementary to such miRNAs into a promoter-luciferase constructincorporated into CFPAC-1 cells (e.g. a CMV promoter-firefly luciferaseconstruct) such that active luciferase will not be expressed in theepithelial state, induction of EMT can readily be monitored by anincrease in luciferase expression or activity. Similarly, in any of theother methods described herein involving a protein that is induciblyexpressed and stimulates an epithelial to mesenchymal transition inCFPAC-1 cells, the biomarker whose level is indicative of the EMT statusof the sample tumor cells can be any of the examples described above.

In the above methods the biomarker whose level is indicative of the EMTstatus of the sample tumor cells is for example a mesenchymal cellbiomarker, e.g. vimentin, fibronectin, N-cadherin, zeb1, twist, FOXC2 orsnail, or the activity of a mesenchymal biomarker gene promoter. In oneembodiment, the activity of a mesenchymal biomarker gene promoter may beassessed by inclusion of a human mesenchymal biomarker genepromoter-reporter gene construct in the engineered CFPAC-1 cells suchthat said promoter reporter activity can be monitored by reporter geneexpression level or activity. In one embodiment, the mesenchymalbiomarker gene promoter-reporter gene construct is a human vimentinpromoter-firefly luciferase construct. Additional examples ofmesenchymal promoters that may be used include the promoters from thefollowing human genes: S100A4 (i.e. S100 calcium binding protein A4(a.k.a. FSP1), GeneID: 6275), SPARC (i.e. secreted protein, acidic,cysteine-rich (osteonectin), GeneID: 6678), IL-11 (i.e. interleukin 11,GeneID: 3589), PCOLCE2 (i.e. procollagen C-endopeptidase enhancer 2,GeneID: 26577), COL6A2 (i.e. collagen, type VI, alpha 2, GeneID: 1292),TFPI2 (i.e. tissue factor pathway inhibitor 2), GeneID: 7980), FBN1(i.e. fibrillin 1, GeneID: 2200), Zeb1 (i.e. zinc finger E-box bindinghomeobox 1, GeneID: 6935), and CHST2 (i.e. carbohydrate(N-acetylglucosamine-6-O) sulfotransferase 2, GeneID: 9435). In analternative embodiment, RNA transcript splicing mechanisms that areunique to human mesenchymal cells (Savagner, P. et al. (1994) Mol BiolCell. 5(8):851-862; Oltean, S. et al. (2006) Proc Natl Acad Sci USA.103(38):14116-14121; Ghigna, C. et al. (2005) Mol. Cell. 20(6):881-890;Bonano, V. I. et al. (2007) Nat. Protoc. 2(9):2166-2181), and do notoperate prior to EMT, in epithelial cells, can be utilized as markers ofEMT status. For example, by including the sequences necessary for suchmesenchymal-specific splicing into a promoter-luciferase constructincorporated into CFPAC-1 cells (e.g. a CMV promoter-firefly luciferaseconstruct) such that active luciferase will only be expressed in themesenchymal state, induction of EMT can readily be monitored by anincrease in luciferase expression or activity. In an alternativeembodiment, miRNAs that are expressed specifically in human mesenchymalcells (e.g. see Hurteau, G. J. et al. (2007) Cancer Research67:7972-7976; Christoffersen, N. R. et al. (2007) RNA 13:1172-1178;Shell, S. et al (2007) Proc Natl. Acad. Sci. 104(27):11400-11405), thatdegrade or diminish translation of transcripts containing complementarynucleic acid sequences, can be utilized as markers of EMT status. Forexample, by including sequences complementary to such miRNAs into apromoter-luciferase construct incorporated into CFPAC-1 cells (e.g. aCMV promoter-firefly luciferase construct) such that active luciferasewill not be expressed in the mesenchymal state, induction of EMT canreadily be monitored by a decrease in luciferase expression or activity.Similarly, in any of the other methods described herein involving aprotein that is inducibly expressed and stimulates an epithelial tomesenchymal transition in CFPAC-1 cells, the biomarker whose level isindicative of the EMT status of the sample tumor cells can be any of theexamples described above.

In any of the methods or cell preparations described herein involving abiomarker gene promoter-reporter gene construct in the engineeredCFPAC-1 cells for monitoring biomarker promoter activity, more than onebiomarker gene promoter-reporter gene construct may be employed so thatmultiple biomarkers may be simultaneously monitored in order to assessEMT status. For example, in one embodiment an epithelial biomarker genepromoter-reporter gene construct and a mesenchymal biomarker genepromoter-reporter gene construct are both used such that decreases inepithelial biomarker gene promoter activity and increases in mesenchymalbiomarker gene promoter activity can both be monitored during EMT. Forexample, the epithelial biomarker gene promoter-reporter gene constructmay be an E-cadherin promoter-firefly luciferase construct and themesenchymal biomarker gene promoter-reporter gene construct may be avimentin promoter-renilla luciferase construct. By using two differentreporter genes that can be independently monitored (e.g. two luciferasesthat produce products that take part in luminescent reactions involvingthe emission of light of different characteristic wavelengths; e.g. seeHawkins, E. H. et al. (2002) Dual-Glo™ Luciferase Assay System:Convenient dual-reporter measurements in 96- and 384-well plates.Promega Notes 81, 22-6; Nieuwenhuijsen B W. et al. (2004) J BiomolScreen. 8, 676-84), both promoters can be monitored simultaneously.Similarly, two or more of the biomarkers described herein aboveinvolving epithelial or mesenchymal specific miRNAs or splicingmechanisms can be simultaneously monitored by using two differentreporter genes that can be independently monitored.

In any of the methods or cell preparations described herein involving abiomarker gene promoter-reporter gene construct in the engineeredCFPAC-1 cells for monitoring biomarker promoter activity by assessingreporter gene expression level, the reporter gene can be anyheterologous gene that expresses a protein whose level is readilydetermined by measuring expressed protein or enzymic activity. Suitablereporter genes include firefly (Photinus pyralis) luciferase, Renilla(Renilla reniformis) luciferase, Gaussia (Gaussia princeps) luciferase,green fluorescent protein (GFP), red fluorescent protein (RFP), etc.(e.g. see Hawkins, E. H. et al. (2002) Dual-Glo™ Luciferase AssaySystem: Convenient dual-reporter measurements in 96- and 384-wellplates. Promega Notes 81, 22-6; Nieuwenhuijsen B W. et al. (2004) J.Biomol. Screen. 8, 676-84; Verhaegen M. and Christopoulos T. K. (2002)Anal. Chem., 74:4378-4385; Tannous, B. A., et al. (2005) Mol. Ther.,11:435-443; Hoffmann, R. M. (2004) Acta Histochemica 106(2):77-87);Hoffmann, R. M. (2008) Methods in Cell Biol. 85:485-495). Gaussialuciferase is a protein that is secreted from cells where it isexpressed, hence it potentially allows, in any of the methods of theinvention, the monitoring of reporter activity secreted into in thegrowth medium of cells in culture, or into the blood, or otherbiological fluids, from cells growing in vivo (e.g. tumor xenografts).

In one embodiment of the above methods, the sample of cells of theepithelial tumor cell line CFPAC-1, which have been engineered toinducibly express a protein that stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells, is an in vivo sample, such as, for example,a xenograft growing in an animal (e.g. immunocompromised mice or rats).Similarly, in any of the other methods or cell preparations describedherein involving a protein that is inducibly expressed and stimulates anepithelial to mesenchymal transition in CFPAC-1 cells, the sample ofcells of the epithelial tumor cell line CFPAC-1 can be an in vivosample, as described above.

The present invention also provides a method of identifying an agentthat inhibits tumor cells that have undergone an epithelial tomesenchymal transition, comprising: contacting a sample of cells of theepithelial tumor cell line CFPAC-1, which have been engineered toinducibly express a protein that stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells, with a compound that induces the expressionof said protein such that an epithelial-to-mesenchymal transition isinduced in the cells, contacting the sample of cells with a test agentto be screened, determining whether the test agent inhibitsmesenchymal-like CFPAC-1 cell growth, and thus determining whether it isan agent that inhibits the growth of tumor cells that have undergone anepithelial to mesenchymal transition. An alternative embodiment of thismethod comprises, after the step of determining whether the test agentinhibits the growth of tumor cells that have undergone an epithelial tomesenchymal transition, the additional steps of determining whether anagent that inhibits mesenchymal-like CFPAC-1 tumor cell growth, alsoinhibits epithelial CFPAC-1 tumor cell growth, and thus determiningwhether it is an agent that specifically inhibits the growth of tumorcells that have undergone an epithelial to mesenchymal transition. In anembodiment of the above methods, an agent that inhibits the growth oftumor cells that have undergone an epithelial to mesenchymal transitionis determined to do so by stimulating apoptosis of said tumor cells. Inanother embodiment of the above methods, an agent that inhibits thegrowth of tumor cells that have undergone an epithelial to mesenchymaltransition is determined to do so by inhibiting proliferation of saidtumor cells.

The present invention also provides a method of identifying an agentthat stimulates mesenchymal-like tumor cells to undergo a mesenchymal toepithelial transition, comprising contacting a sample of cells of theepithelial tumor cell line CFPAC-1, which have been engineered toinducibly express a protein that stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells, with a compound that induces the expressionof said protein such that an epithelial-to-mesenchymal transition isinduced in the cells, contacting the sample of cells with a test agentto be screened, determining whether the test agent stimulates themesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal toepithelial transition, by comparing the level of a biomarker whose levelis indicative of the EMT status of the sample tumor cells to the levelof the same biomarker in an identical sample of mesenchymal-like CFPAC-1cells not contacted with the test agent, and thus determining whetherthe test agent is an agent that stimulates mesenchymal-like tumor cellsto undergo a mesenchymal to epithelial transition.

The present invention also provides a tumor cell preparation for use inthe identification of anti-cancer agents, wherein said tumor cellpreparation comprises: a sample of cells of the epithelial tumor cellline CFPAC-1, which have been engineered to inducibly express a proteinthat stimulates an epithelial to mesenchymal transition in CFPAC-1cells. In one embodiment, the tumor cell preparation also comprises amesenchymal biomarker gene promoter-reporter gene construct in theengineered CFPAC-1 cells so that the activity of the mesenchymalbiomarker gene promoter can be assessed by monitoring reporter genelevel or activity. In one embodiment, the mesenchymal biomarker genepromoter-reporter gene construct is a vimentin promoter-fireflyluciferase construct. In another embodiment, the tumor cell preparationcomprises a epithelial biomarker gene promoter-reporter gene constructin the engineered CFPAC-1 cells so that the activity of the epithelialbiomarker gene promoter can be assessed by monitoring reporter genelevel or activity. In one embodiment, the epithelial biomarker genepromoter-reporter gene construct is an E-cadherin promoter-fireflyluciferase construct. In the above cell preparations the protein that isinducibly expressed and stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells can be any of those identified for use inthe methods described herein above that utilize such a cell preparation(e.g. Snail, Zeb1 etc). In one embodiment of this cell preparation, aTet-regulated promoter is used to inducibly express (e.g. usingdoxycyclin, or tetracycline) the protein that stimulates an epithelialto mesenchymal transition in the CFPAC-1 cells. In the aboveembodiments, the biomarker gene promoter-reporter gene construct isstably expressed by the engineered CFPAC-1 cell. Accordingly, thisinvention provides, for use in the identification of anti-cancer agents,both an epithelial CFPAC-1 tumor cell preparation, prior to induction ofa protein that stimulates an epithelial to mesenchymal transition, and amesenchymal-like tumor cell preparation, after induction of a proteinthat stimulates an epithelial to mesenchymal transition. As describedherein, these cell preparations can be used for screening test agents toidentify agents that inhibit either cell type, or to find agents thatwill inhibit EMT or stimulate MET.

In the context of the methods of this invention, epithelial ormesenchymal biomarkers expressed by a tumor cell can include molecularand cellular markers that indicate the transition state of the tumorcell. In a preferred embodiment the biomarker is an individual markerprotein, or its encoding mRNA, characteristic of the particulartransition state of the tumor cell, i.e. a tumor cell exhibitingepithelial or mesenchymal characteristics. In an alternative embodiment,in certain circumstances the biomarker may be a characteristicmorphological pattern produced in the tumor cell by cellularmacromolecules that is characteristic of either an epithelial ormesenchymal condition. Thus, morphometric cell analysis can be used toprovide information on epithelial or mesenchymal status of tumor cells.In an additional embodiment the biomarker that indicates the transitionstate of the tumor cell is methylation of the E-Cadherin gene (CDH1)promoter. CDH1 promoter methylation indicates that tumor cells haveundergone an EMT transition.

TABLE 1 Molecular Biomarker Gene Identification Human Biomarker NCBIGeneID¹ NCBI RefSeq² E-cadherin 999 NP_004351 Brk 5753 NP_005966γ-catenin 3728 NP_002221 α1-catenin 1495 NP_001894 α2-catenin 1496NP_004380 α3-catenin 29119 NP_037398 keratin 8 3856 NP_002264 keratin 183875 NP_000215 vimentin 7431 NP_003371 fibronectin 1 2335 NP_002017fibrillin-1 2200 NP_000129 fibrillin-2 2201 NP_001990 collagenalpha2(IV) 1284 NP_001837 collagen alpha2(V) 1290 NP_000384 LOXL1 4016NP_005567 nidogen 4811 NP_002499 C11orf9 745 NP_037411 tenascin 3371NP_002151 N-cadherin 1000 NP_001783 ¹The NCBI GeneID number is a uniqueidentifier of the biomarker gene from the NCBI Entrez Gene databaserecord (National Center for Biotechnology Information (NCBI), U.S.National Library of Medicine, 8600 Rockville Pike, Building 38A,Bethesda, MD 20894; Internet address http://www.ncbi.nlm.nih.gov/). ²TheNCBI RefSeq (Reference Sequence) is an example of a sequence expressedby the biomarker gene.

TABLE 2 Molecular Biomarker Gene Identification Human Biomarker³ NCBIGeneID¹ NCBI RefSeq² Epithelial keratin K1 3848 NP_006112 keratin K23849 NP_000414 keratin K3 3850 NP_476429 keratin K4 3851 NP_002263keratin K5 3852 NP_000415 keratin K6a 3853 NP_005545 keratin K6b 3854NP_005546 keratin K6c 286887 NP_775109 keratin K7 3855 NP_005547 keratinK8 3856 NP_002264 keratin K9 3857 NP_000217 keratin K10 3858 NP_000412keratin K12 3859 NP_000214 keratin K13 3860 NP_002265 keratin K14 3861NP_000517 keratin K15 3866 NP_002266 keratin K16 3868 NP_005548 keratinK17 3872 NP_000413 keratin K18 3875 NP_000215 keratin K19 3880 NP_002267keratin K20 54474 NP_061883 keratin K23 25984 NP_056330 keratin K24192666 NP_061889 keratin K25 147183 NP_853512 keratin K26 353288NP_853517 keratin K27 342574 NP_853515 keratin K28 162605 NP_853513keratin K71 112802 NP_258259 keratin K72 140807 NP_542785 keratin K73319101 NP_778238 keratin K74 121391 NP_778223 keratin K75 9119 NP_004684keratin K76 51350 NP_056932 keratin K77 374454 NP_778253 keratin K78196374 NP_775487 keratin K79 338785 NP_787028 keratin K80 144501NP_001074961 Mesenchymal vimentin 7431 NP_003371 ¹The NCBI GeneID numberis a unique identifier of the biomarker gene from the NCBI Entrez Genedatabase record (National Center for Biotechnology Information (NCBI),U.S. National Library of Medicine, 8600 Rockville Pike, Building 38A,Bethesda, MD 20894; Internet address http://www.ncbi.nlm.nih.gov/). ²TheNCBI RefSeq (Reference Sequence) is an example of a sequence expressedby the biomarker gene. ³The new consensus nomenclature has been usedherein when referring to keratins (see Schweizer, J. et al. (2006) J.Cell Biol. 174(2): 169-174). Former names for these proteins can befound in the latter reference, and at the Human Intermediate FilamentDatabase (http://www.interfil.org/index.php). N.B. Epithelial keratinsor cytokeratins are intermediate filament keratins. The terms “keratin”and “cytokeratin” are used synonymously herein. When refering tospecific keratins in the text herein the “K” in the standard keratindesignation (as in Table 2) is generally dropped (e.g. keratin K8 =keratin 8).

Table 1 lists genes coding for examples of epithelial or mesenchymalmolecular biomarkers that can be used in the practice of the methods ofthe invention described herein. The epithelial or mesenchymal molecularbiomarkers can include any product expressed by these genes, includingvariants thereof, e.g. expressed mRNA or protein, splice variants, co-and post-translationally modified proteins, polymorphic variants etc. Inone embodiment the biomarker is the embryonal EDB⁺ fibronectin, a splicevariant expressed by the fibronectin 1 gene (Kilian, O. et al. (2004)Bone 35(6):1334-1345). A possible advantage of determining this fetalform of fibronectin is that one could readily distinguishmesenchymal-like tumors from surrounding stromal tissue. Table 2 listsgenes coding for examples of molecular biomarkers that can be used inthe practice of certain embodiments of the methods of the inventiondescribed herein wherein co-expression of epithelial keratin(s) and themesenchymal biomarker vimentin at similar levels is indicative of amesenchymal-like cell. The molecular biomarkers can include any productexpressed by these genes, including variants thereof, e.g. expressedmRNA or protein, splice variants, co- and post-translationally modifiedproteins, polymorphic variants etc.

In another embodiment of any of the methods of the invention describedherein, the mesenchymal biomarker utilized in the method is selectedfrom the human transcriptional repressors Snail (NCBI GeneID 6615), Zeb1(NCBI GeneID 6935), Twist (NCBI GeneID 7291), Sip 1 NCBI GeneID 8487),and Slug (NCBI GeneID 6591).

In another embodiment of the methods of this invention the mesenchymalbiomarker is methylation of the promoter of a gene whose transcriptionis repressed as a result of EMT in the tumor cell. In the context ofthis method high levels of a tumor cell mesenchymal biomarkeressentially means readily detectable methylation of the promoter (e.g. astrong signal during detection of a methylation-specific PCR-amplifiednucleic acid product derived from a promoter methylation site), whereaslow levels of a tumor cell mesenchymal biomarker essentially means nodetectable or low methylation of the promoter (e.g. no, or acomparatively weak, signal during detection of a methylation-specificPCR-amplified nucleic acid product derived from a promoter methylationsite). In one embodiment of this method the gene whose transcription isrepressed as a result of EMT in the tumor cell is the E-Cadherin gene(i.e. CDH1; NCBI GeneID 999). In another embodiment of this method thegene whose transcription is repressed as a result of EMT in the tumorcell is the γ-catenin gene (i.e. NCBI GeneID 3728). In anotherembodiment of this method the gene whose transcription is repressed as aresult of EMT in the tumor cell is an α-catenin gene (e.g. NCBI GeneID1495, 1496, or 29119). In another embodiment of this method the genewhose transcription is repressed as a result of EMT in the tumor cell isa cytokeratin gene (e.g. NCBI GeneID 3856 (keratin 8) or 3875 (keratin18)).

Examples of additional epithelial markers that can be used in any of themethods of this invention include phospho-14-3-3 epsilon, 14-3-3 gamma(KCIP-1), 14-3-3 sigma (Stratifin), 14-3-3 zeta/delta,phospho-serine/threonine phosphatase 2A, 4F2hc(CD98 antigen), adeninenucleotide translocator 2, annexin A3, ATP synthase beta chain,phospho-insulin receptor substrate p53/p54, Basigin (CD147 antigen),phospho-CRK-associated substrate (p130Cas), Bcl-X, phospho-P-cadherin,phospho-calmodulin (CaM), Calpain-2 catalytic subunit, Cathepsin D,Cofilin-1, Calpain small subunit 1, Catenin beta-1, Catenin delta-1 (p120 catenin), Cystatin B, phospho-DAZ-associated protein 1, Carbonylreductase [NADPH], Diaphanous-related formin 1 (DRF1), Desmoglein-2,Elongation factor 1-delta, phospho-p185erbB2, Ezrin (p81), phospho-focaladhesion kinase 1, phospho-p94-FER (c-FER)., Filamin B,phospho-GRB2-associated binding protein 1, Rho-GDI alpha, phospho-GRB2,GRP 78, Glutathione S-transferase P, 3-hydroxyacyl-CoA dehydrogenase,HSP 90-alpha, HSP70.1, eIF3 p110, eIF-4E, Leukocyte elastase inhibitor,Importin-4, Integrin alpha-6, Integrin beta-4, phospho-Cytokeratin 17,Cytokeratin 19, Cytokeratin 7, Casein kinase I, alpha, Protein kinase C,delta, Pyruvate kinase, isozymes M1/M2, phospho-Erbin, LIM and SH3domain protein 1 (LASP-1), 4F21c (CD98 light chain), L-lactatedehydrogenase A chain, Galectin-3, Galectin-3 binding protein,phospho-LIN-7 homolog C, MAP (APC-binding protein EB1), Maspin precursor(Protease inhibitor 5), phospho-Met tyrosine kinase (HGF receptor),Mixed-lineage leukemia protein 2, Monocarboxylate transporter 4,phospho-C-Myc binding protein (AMY-1), Myosin-9, Myosin lightpolypeptide 6, Nicotinamide phosphoribosyltransferase, Niban-likeprotein (Meg-3), Ornithine aminotransferase, phospho-Occludin, Ubiquitinthiolesterase, PAF acetylhydrolase IB beta subunit,phospho-partitioning-defective 3 (PAR-3), phospho-programmed cell death6-interacting protein, phospho-Programmed cell death protein 6, Proteindisulfide-isomerase, phospho-plakophilin-2, phospho-plakophilin-3,Protein phosphatase 1, Peroxiredoxin 5, Proteasome activator complexsubunit 1, Prothymosin alpha, Retinoic acid-induced protein 3,phospho-DNA repair protein REV 1, Ribonuclease inhibitor, RuvB-like 1,S-100P, S-100L, Calcyclin, S100C, phospho-Sec23A, phospho-Sec23B,Lysosome membrane protein II (LIMP II), p60-Src, phospho-Amplaxin(EMS1), SLP-2, Gamma-synuclein, Tumor calcium signal transducer 1, Tumorcalcium signal transducer 2, Transgelin-2, Transaldolase, Tubulin beta-2chain, Translationally controlled (TCTP), Tissue transglutaminase,Transmembrane protein Tmp21, Ubiquitin-conjugating enzyme E2 N,UDP-glucosyltransferase 1, phospho-p6′-Yes, phospho-Tight junctionprotein ZO-1, AHNAK (Desmoyokin), phospho-ATP synthase beta chain,phospho-ATP synthase delta, Cold shock domain protein E1, DesmoplakinIII, Plectin 1, phospho-Nectin 2 (CD112 antigen), phospho-p185-Ron,phospho-SHC1, E-cadherin, Brk, γ-catenin, α1-catenin, α2-catenin,α3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin1, laminin alpha-5, ST14, and other epithelial biomarkers known in theart (see for example, US Patent Application Publication 2007/0212738;U.S. Patent Application 60/923,463; U.S. Patent Application 60/997,514).Where the epithelial biomarker is a phospho-“protein” the extent ofphosphorylation of the protein rather than the level of the protein perse is the parameter that is altered after EMT. The altered level ofphosphorylation of these proteins is also understood to be due tochanges in the level of phosphorylation of one or more tyrosine residuesof the protein (US Patent Application Publication 2007/0212738).

Examples of additional mesenchymal markers that can be used in any ofthe methods of this invention include MMP9 (matrix-metalloproteinase 9;NCBI Gene ID No. 4318), MHC class I antigen A*1, Acyl-CoA desaturase,LANP-like protein (LANP-L), Annexin A6, ATP synthase gamma chain,BAG-family molecular chaperone regulator-2, phospho-Bullous pemphigoidantigen, phospho-Protein Clorf77, CDK1 (cdc2), phospho-Clathrin heavychain 1, Condensin complex subunit 1,3,2-trans-enoyl-CoA isomerase,DEAH-box protein 9, phospho-Enhancer of rudimentary homolog,phospho-Fibrillarin, GAPDH muscle, GAPDH liver, Synaptic glycoproteinSC2, phospho-Histone H1.0, phospho-Histone H1.2, phospho-Histone H1.3,phospho-Histone H1.4, phospho-Histone H1.5, phospho-Histone H1x,phospho-Histone H2AFX, phospho-H1stone H2A.o, phospho-H1stone H2A.q,phospho-Histone H2A.z, phospho-Histone H2B.j, phospho-Histone H2B.r,phospho-Histone H4, phospho-HMG-17-like 3, phospho-HMG-14,phospho-HMG-17, phospho-HMGI-C, phospho-HMG-I/HMG-Y, phospho-Thyroidreceptor interacting protein 7 (TRIP7), phospho-hnRNP H3, hnRNP C1/C2,hnRNP F, phospho-hnRNP G, eIF-5A, NFAT 45 kDa, Importin beta-3,cAMP-dependent PK1a, Lamin B1, Lamin A/C, phospho-Laminin alpha-3 chain,L-lactate dehydrogenase B chain, Galectin-1, phospho-Fez1,Hyaluronan-binding protein 1, phospho-Microtubule-actin crosslinkingfactor 1, Melanoma-associated antigen 4, Matrin-3, Phosphate carrierprotein, Myosin-10, phospho-N-acylneuraminate cytidylyltransferase,phospho-NHP2-like protein 1, H/ACA ribonucleoprotein subunit 1,Nucleolar phosphoprotein p130, phospho-RNA-binding protein Nova-2,Nucleophosmin (NPM), NADH-ubiquinone oxidoreductase 39 kDa subunit,phospho-Polyadenylate-binding protein 2, Prohibitin, Prohibitin-2,Splicing factor Prp8, Polypyrimidine tract-binding protein 1,Parathymosin, Rab-2A, phospho-RNA-binding protein Raly, PutativeRNA-binding protein 3, phospho-60S ribosomal protein L23, hnRNP A0,hnRNP A2/B1, hnRNP A/B, U2 small nuclear ribonucleoprotein B,phospho-Ryanodine receptor 3, phospho-Splicing factor 3A subunit 2,snRNP core protein D3, Nesprin-1, Tyrosine—tRNA ligase,phospho-Tankyrase 1-BP, Tubulin beta-3, Acetyl-CoA acetyltransferase,phospho-bZIP enhancing factor BEF (Aly/REF; Tho4), Ubiquitin, Ubiquitincarboxyl-terminal hydrolase 5, Ubiquinol-cytochrome c reductase,Vacuolar protein sorting 16, phospho-Zinc finger protein 64,phospho-AHNAK (Desmoyokin), ATP synthase beta chain, ATP synthase deltachain, phospho-Cold shock domain protein E1, phospho-Plectin 1, Nectin 2(CD112 antigen), p185-Ron, SHC1, vimentin, fibronectin, fibrillin-1,fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen,C11orf9, tenascin, N-cadherin, embryonal EDB⁺ fibronectin, tubulinalpha-3, epimorphin, and other mesenchymal biomarkers known in the art,(see for example, US Patent Application Publication 2007/0212738; U.S.Patent Application 60/923,463; U.S. Patent Application 60/997,514).Where the mesenchymal biomarker is a phospho-“protein” the extent ofphosphorylation of the protein rather than the level of the protein perse is the parameter that is altered after EMT. The altered level ofphosphorylation of these proteins is also understood to be due tochanges in the level of phosphorylation of one or more tyrosine residuesof the protein (US Patent Application Publication 2007/0212738).

The biomarkers in the above lists of epithelial and mesenchymalbiomarkers have been identified as being altered in expression level (orphosphoylation level for phospho-“proteins”) after EMT (see for example,US Patent Application Publication 2007/0212738, the contents of whichare incorporated herein by reference; US Published Application2006/0211060 (filed Mar. 16, 2006); Thomson, S. et al. (2005) CancerRes. 65(20) 9455-9462; and Yauch, R. L. et al. (2005) Clin. Can. Res.11(24) 8686-8698).

In the methods of this invention, biomarker expression level can beassessed relative to a control molecule whose expression level remainsconstant throughout EMT or when comparing tumor cells expressing eitherepithelial or mesenchymal transition states as indicated by molecularbiomarkers (e.g. a “housekeeping” gene, such as GAPDH, β-actin, tubulin,or the like). Biomarker expression level can also be assessed relativeto the other type of tumor cell biomarker (i.e. epithelial compared tomesenchymal), or to the biomarker level in non-tumor cells of the sametissue, or another cell or tissue source used as an assay reference.

In the methods of this invention, the level of an epithelial ormesenchymal biomarker expressed by a tumor cell can be assessed by usingany of the standard bioassay procedures known in the art fordetermination of the level of expression of a gene, including forexample ELISA, RIA, immunopreciptation, immunoblotting,immunofluorescence microscopy, immunohistochemistry (IHC), RT-PCR, insitu hybridization, cDNA microarray, or the like, as described in moredetail below. In an embodiment of any of these methods, their use iscoupled with a method to isolate a particular cell population, e.g.laser capture microdissection (LCM). In an additional embodiment, FACSanalysis can be used with immunofluorescence biomarker (e.g. E-cadherin)labeling to isolate and quantify cell populations expressing differentepithelial or mesenchymal biomarkers, and thus for example thepercentage of cells that have undergone an EMT can be estimated (e.g.see Xu, Z. et al. (2003) Cell Research 13(5):343-350).

In the methods of this invention, the expression level of a tumor cellepithelial or mesenchymal biomarker in vivo is preferably assessed byassaying a tumor biopsy. In one embodiment the biopsy comprises samplestaken from multiple areas of the tumor, or a method (e.g. core needlebiopsy) that samples different areas of the tumor, thus ensuring thatwhen the tumor is of a heterogeneous nature with respect to the types ofcells it contains, that a representative biopsy is obtained. In analternative embodiment, given that a tumor may be heterogeneous withrespect to the EMT status of the cells it contains, the methods of thisinvention are preferably applied separately to different cell types(e.g. using IHC, or an analysis method coupled with a step to isolate aparticular cell population). Alternatively, by employing cell surfaceepithelial and/or mesenchymal biomarker antibodies (e.g. to E-cadherin),FACS analysis can be used to isolate and quantify the numbers of tumorcells at different stages of EMT.

However, in an alternative embodiment, expression level of the tumorcell biomarker can be assessed in bodily fluids or excretions containingdetectable levels of biomarkers originating from the tumor or tumorcells. Bodily fluids or excretions useful in the present inventioninclude blood, urine, saliva, stool, pleural fluid, lymphatic fluid,sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or anyother bodily secretion or derivative thereof. By blood it is meant toinclude whole blood, plasma, serum or any derivative of blood.Assessment of tumor epithelial or mesenchymal biomarkers in such bodilyfluids or excretions can sometimes be preferred in circumstances wherean invasive sampling method is inappropriate or inconvenient.

For assessment of tumor cell epithelial or mesenchymal biomarkerexpression, tumor samples containing tumor cells, or proteins or nucleicacids produced by these tumor cells, may be used in the methods of thepresent invention. In these embodiments, the level of expression of thebiomarker can be assessed by assessing the amount (e.g. absolute amountor concentration) of the marker in a tumor cell sample, e.g., a tumorbiopsy obtained from an animal, or another sample containing materialderived from the tumor (e.g. blood, serum, urine, or other bodily fluidsor excretions as described herein above). The cell sample can, ofcourse, be subjected to a variety of well-known post-collectionpreparative and storage techniques (e.g., nucleic acid and/or proteinextraction, fixation, storage, freezing, ultrafiltration, concentration,evaporation, centrifugation, etc.) prior to assessing the amount of themarker in the sample. Likewise, tumor biopsies may also be subjected topost-collection preparative and storage techniques, e.g., fixation.

Determination of epithelial or mesenchymal biomarker levels in in vivostudies can be assessed by a number of different approaches, includingdirect analysis of proteins that segregate as epithelial related (e.g.E-cadherin) or mesenchymal related (e.g. vimentin, Zeb1) biomarkers. Anadvantage of this approach is that EMT markers are read directly, andthe relative amounts of cell populations expressing epithelial ormesenchymal biomarkers can readily be examined and quantified, by forexample FACS analysis (e.g. see Xu, Z. et al. (2003) Cell Research13(5):343-350). However, this approach also requires sufficientquantities of cells or tissue in order to perform an analysis (e.g.immunohistochemistry). Sufficient quantities of tissue may be difficultto obtain from certain procedures such as FNA (fine needle aspiration).Core biopsies provide larger amounts of tissue, but are sometimes notreadily available. Alternatively, these EMT biomarkers could beevaluated based upon the expression level of their encoding RNAtranscripts using a quantitative PCR based approach. An advantage ofthis approach is that very few tumor cells are required for thismeasurement, and it is very likely that sufficient material may beobtained via an FNA. However, here the transcript levels for a givenbiomarker may be derived from both tumor cells as well as infiltratingstromal cells from the tumor. Given that stromal cells also expressmesenchymal cell markers, this may obscure detection of the EMT statusfor tumor cells. Use of in situ hybridization (e.g. FISH) or tissuemicrodisection may be useful here to overcome this potential limitation.

Given that the expression level of E-cadherin is a hallmark of the EMTstatus for a tumor cell, EMT may also be evaluated based upon themethylation status of the E-cadherin promoter, as described herein.Methylation silences transcription, and so a high level of methylationcorrelates with a mesenchymal-like state. A potential benefit of thisapproach is that, like measurement of transcript levels, measuring themethylation status of DNA would likely require very little material.Sufficient material could likely be obtained from an FNA and would notrequire a core biopsy. Additionally, since this approach involvesevaluation of DNA and not RNA, it is likely to be a more stable read-outover time, such as during medium or long term storage of a sample.

In the methods of the invention, one can detect expression of biomarkerproteins having at least one portion which is displayed on the surfaceof tumor cells which express it. It is a simple matter for the skilledartisan to determine whether a marker protein, or a portion thereof, isexposed on the cell surface. For example, immunological methods may beused to detect such proteins on whole cells, or well knowncomputer-based sequence analysis methods may be used to predict thepresence of at least one extracellular domain (i.e. including bothsecreted proteins and proteins having at least one cell-surface domain).Expression of a marker protein having at least one portion which isdisplayed on the surface of a cell which expresses it may be detectedwithout necessarily lysing the tumor cell (e.g. using a labeled antibodywhich binds specifically with a cell-surface domain of the protein).

Expression of a biomarkers described in this invention may be assessedby any of a wide variety of well known methods for detecting expressionof a transcribed nucleic acid or protein. Non-limiting examples of suchmethods include immunological methods for detection of secreted,cell-surface, cytoplasmic, or nuclear proteins, protein purificationmethods, protein function or activity assays, nucleic acid hybridizationmethods, nucleic acid reverse transcription methods, and nucleic acidamplification methods.

In one embodiment, expression of a biomarker is assessed using anantibody (e.g. a radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative(e.g. an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair {e.g. biotin-streptavidin}), or anantibody fragment (e.g. a single-chain antibody, an isolated antibodyhypervariable domain, etc.) which binds specifically with a biomarkerprotein or fragment thereof, including a biomarker protein which hasundergone either all or a portion of post-translational modifications towhich it is normally subjected in the tumor cell (e.g. glycosylation,phosphorylation, methylation etc.).

In another embodiment, expression of a biomarker is assessed bypreparing mRNA/cDNA (i.e. a transcribed polynucleotide) from tumorcells, and by hybridizing the mRNA/cDNA with a reference polynucleotidewhich is a complement of a biomarker nucleic acid, or a fragment thereofcDNA can, optionally, be amplified using any of a variety of polymerasechain reaction methods prior to hybridization with the referencepolynucleotide. Expression of one or more biomarkers can likewise bedetected using quantitative PCR to assess the level of expression of thebiomarker(s). Alternatively, any of the many known methods of detectingmutations or variants (e.g. single nucleotide polymorphisms, deletions,etc.) of a biomarker of the invention may be used to detect occurrenceof a biomarker in a tumor cell.

In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g. at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or morenucleotide residues) of a biomarker nucleic acid. If polynucleotidescomplementary to or homologous with are differentially detectable on thesubstrate (e.g. detectable using different chromophores or fluorophores,or fixed to different selected positions), then the levels of expressionof a plurality of biomarkers can be assessed simultaneously using asingle substrate (e.g. a “gene chip” microarray of polynucleotides fixedat selected positions). When a method of assessing biomarker expressionis used which involves hybridization of one nucleic acid with another,it is preferred that the hybridization be performed under stringenthybridization conditions.

When a plurality of biomarkers of the invention are used in the methodsof the invention, the level of expression of each biomarker in tumorcells induced to undergo EMT can be compared with the level ofexpression of each of the plurality of biomarkers in non-induced samplesof the same type, either in a single reaction mixture (i.e. usingreagents, such as different fluorescent probes, for each biomarker) orin individual reaction mixtures corresponding to one or more of thebiomarkers.

An exemplary method for detecting the presence or absence of a biomarkerprotein or nucleic acid in a biological sample involves obtaining abiological sample (e.g. a tumor-associated body fluid) from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting the polypeptide or nucleic acid (e.g., mRNA,genomic DNA, or cDNA). The detection methods of the invention can thusbe used to detect mRNA, protein, cDNA, or genomic DNA, for example, in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of mRNA include Northern hybridizations and insitu hybridizations. In vitro techniques for detection of a biomarkerprotein include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of genomic DNA include Southern hybridizations. In vivotechniques for detection of mRNA include polymerase chain reaction(PCR), Northern hybridizations and in situ hybridizations. Furthermore,in vivo techniques for detection of a biomarker protein includeintroducing into a subject a labeled antibody directed against theprotein or fragment thereof. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain a biomarker, anda probe, under appropriate conditions and for a time sufficient to allowthe biomarker and probe to interact and bind, thus forming a complexthat can be removed and/or detected in the reaction mixture. Theseassays can be conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoringthe biomarker or probe onto a solid phase support, also referred to as asubstrate, and detecting target biomarker/probe complexes anchored onthe solid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of biomarker, can be anchored onto a carrier orsolid phase support. In another embodiment, the reverse situation ispossible, in which the probe can be anchored to a solid phase and asample from a subject can be allowed to react as an unanchored componentof the assay.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, biomarker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which thebiomarker or probe belongs. Well-known supports or carriers include, butare not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of biomarker/probe complexes anchored tothe solid phase can be accomplished in a number of methods outlinedherein.

In one embodiment, the probe, when it is the unanchored assay component,can be labeled for the purpose of detection and readout of the assay,either directly or indirectly, with detectable labels discussed hereinand which are well-known to one skilled in the art.

It is also possible to directly detect biomarker/probe complex formationwithout further manipulation or labeling of either component (biomarkeror probe), for example by utilizing the technique of fluorescence energytransfer (i.e. FET, see for example, Lakowicz et al., U.S. Pat. No.5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). Afluorophore label on the first, ‘donor’ molecule is selected such that,upon excitation with incident light of appropriate wavelength, itsemitted fluorescent energy will be absorbed by a fluorescent label on asecond ‘acceptor’ molecule, which in turn is able to fluoresce due tothe absorbed energy. Alternately, the ‘donor’ protein molecule maysimply utilize the natural fluorescent energy of tryptophan residues.Labels are chosen that emit different wavelengths of light, such thatthe ‘acceptor’ molecule label may be differentiated from that of the‘donor’. Since the efficiency of energy transfer between the labels isrelated to the distance separating the molecules, spatial relationshipsbetween the molecules can be assessed. In a situation in which bindingoccurs between the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a biomarker can be accomplished without labeling either assaycomponent (probe or biomarker) by utilizing a technology such asreal-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander,S, and Urbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al.,1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or“surface plasmon resonance” is a technology for studying biospecificinteractions in real time, without labeling any of the interactants(e.g., BIAcore). Changes in the mass at the binding surface (indicativeof a binding event) result in alterations of the refractive index oflight near the surface (the optical phenomenon of surface plasmonresonance (SPR)), resulting in a detectable signal which can be used asan indication of real-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with biomarker and probe as solutesin a liquid phase. In such an assay, the complexed biomarker and probeare separated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, biomarker/probe complexes may be separated fromuncomplexed assay components through a series of centrifugal steps, dueto the different sedimentation equilibria of complexes based on theirdifferent sizes and densities (see, for example, Rivas, G., and Minton,A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of thebiomarker/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J. Chromatogr B Biomed SciAppl 1997 Oct. 10; 699(1-2):499-525). Gel electrophoresis may also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically preferred.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

In a particular embodiment, the level of biomarker mRNA can bedetermined both by in situ and by in vitro formats in a biologicalsample using methods known in the art. The term “biological sample” isintended to include tissues, cells, biological fluids and isolatesthereof, isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Many expression detection methods use isolatedRNA. For in vitro methods, any RNA isolation technique that does notselect against the isolation of mRNA can be utilized for thepurification of RNA from tumor cells (see, e.g., Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, New York1987-1999). Additionally, large numbers of tissue samples can readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski(1989, U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding a biomarkerof the present invention. Other suitable probes for use in thediagnostic assays of the invention are described herein. Hybridizationof an mRNA with the probe indicates that the biomarker in question isbeing expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the biomarkers of the present invention.

An alternative method for determining the level of mRNA biomarker in asample involves the process of nucleic acid amplification, e.g., byRT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat.No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad.Sci. USA, 88:189-193), self sustained sequence replication (Guatelli etal., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the tumorcells prior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to mRNA that encodes the biomarker.

As an alternative to making determinations based on the absoluteexpression level of the biomarker, determinations may be based on thenormalized expression level of the biomarker. Expression levels arenormalized by correcting the absolute expression level of a biomarker bycomparing its expression to the expression of a gene that is not abiomarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a tumor cellsample, to another sample, e.g., a non-tumor sample, or between samplesfrom different sources, or between samples before and after induction ofEMT.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of abiomarker (e.g. a mesenchymal biomarker), the level of expression of thebiomarker is determined for 10 or more samples of normal versus cancercell isolates, preferably 50 or more samples, prior to the determinationof the expression level for the sample in question. The mean expressionlevel of each of the genes assayed in the larger number of samples isdetermined and this is used as a baseline expression level for thebiomarker. The expression level of the biomarker determined for the testsample (absolute level of expression) is then divided by the meanexpression value obtained for that biomarker. This provides a relativeexpression level.

In another embodiment of the present invention, a biomarker protein isdetected. A preferred agent for detecting biomarker protein of theinvention is an antibody capable of binding to such a protein or afragment thereof, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment or derivative thereof (e.g., Fab orF(ab′).sub.2) can be used. The term “labeled”, with regard to the probeor antibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin.

Proteins from tumor cells can be isolated using techniques that are wellknown to those of skill in the art. The protein isolation methodsemployed can, for example, be such as those described in Harlow and Lane(Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

A variety of formats can be employed to determine whether a samplecontains a protein that binds to a given antibody. Examples of suchformats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbant assay (ELISA). A skilled artisan can readily adapt knownprotein/antibody detection methods for use in determining whether tumorcells express a biomarker of the present invention.

In one format, antibodies, or antibody fragments or derivatives, can beused in methods such as Western blots or immunofluorescence techniquesto detect the expressed proteins. In such uses, it is generallypreferable to immobilize either the antibody or proteins on a solidsupport. Suitable solid phase supports or carriers include any supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, protein isolated from tumorcells can be run on a polyacrylamide gel electrophoresis and immobilizedonto a solid phase support such as nitrocellulose. The support can thenbe washed with suitable buffers followed by treatment with thedetectably labeled antibody. The solid phase support can then be washedwith the buffer a second time to remove unbound antibody. The amount ofbound label on the solid support can then be detected by conventionalmeans.

For ELISA assays, specific binding pairs can be of the immune ornon-immune type. Immune specific binding pairs are exemplified byantigen-antibody systems or hapten/anti-hapten systems. There can bementioned fluorescein/anti-fluorescein,dinitrophenyl/anti-dinitrophenyl, biotin/anti-biotin,peptide/anti-peptide and the like. The antibody member of the specificbinding pair can be produced by customary methods familiar to thoseskilled in the art. Such methods involve immunizing an animal with theantigen member of the specific binding pair. If the antigen member ofthe specific binding pair is not immunogenic, e.g., a hapten, it can becovalently coupled to a carrier protein to render it immunogenic.Non-immune binding pairs include systems wherein the two componentsshare a natural affinity for each other but are not antibodies.Exemplary non-immune pairs are biotin-streptavidin, intrinsicfactor-vitamin B₁₂, folic acid-folate binding protein and the like.

A variety of methods are available to covalently label antibodies withmembers of specific binding pairs. Methods are selected based upon thenature of the member of the specific binding pair, the type of linkagedesired, and the tolerance of the antibody to various conjugationchemistries. Biotin can be covalently coupled to antibodies by utilizingcommercially available active derivatives. Some of these arebiotin-N-hydroxy-succinimide which binds to amine groups on proteins;biotin hydrazide which binds to carbohydrate moieties, aldehydes andcarboxyl groups via a carbodiimide coupling; and biotin maleimide andiodoacetyl biotin which bind to sulfhydryl groups. Fluorescein can becoupled to protein amine groups using fluorescein isothiocyanate.Dinitrophenyl groups can be coupled to protein amine groups using2,4-dinitrobenzene sulfate or 2,4-dinitrofluorobenzene. Other standardmethods of conjugation can be employed to couple monoclonal antibodiesto a member of a specific binding pair including dialdehyde,carbodiimide coupling, homofunctional crosslinking, andheterobifunctional crosslinking. Carbodiimide coupling is an effectivemethod of coupling carboxyl groups on one substance to amine groups onanother. Carbodiimide coupling is facilitated by using the commerciallyavailable reagent 1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide (EDAC).

Homobifunctional crosslinkers, including the bifunctional imidoestersand bifunctional N-hydroxysuccinimide esters, are commercially availableand are employed for coupling amine groups on one substance to aminegroups on another. Heterobifunctional crosslinkers are reagents whichpossess different functional groups. The most common commerciallyavailable heterobifunctional crosslinkers have an amine reactiveN-hydroxysuccinimide ester as one functional group, and a sulfhydrylreactive group as the second functional group. The most commonsulfhydryl reactive groups are maleimides, pyridyl disulfides and activehalogens. One of the functional groups can be a photoactive arylnitrene, which upon irradiation reacts with a variety of groups.

The detectably-labeled antibody or detectably-labeled member of thespecific binding pair is prepared by coupling to a reporter, which canbe a radioactive isotope, enzyme, fluorogenic, chemiluminescent orelectrochemical materials. Two commonly used radioactive isotopes are¹²⁵I and ³H. Standard radioactive isotopic labeling procedures includethe chloramine T, lactoperoxidase and Bolton-Hunter methods for ¹²⁵I andreductive methylation for ³H. The term “detectably-labeled” refers to amolecule labeled in such a way that it can be readily detected by theintrinsic enzymic activity of the label or by the binding to the labelof another component, which can itself be readily detected.

Enzymes suitable for use in this invention include, but are not limitedto, horseradish peroxidase, alkaline phosphatase, β-galactosidase,glucose oxidase, luciferases, including firefly and renilla,β-lactamase, urease, green fluorescent protein (GFP) and lysozyme.Enzyme labeling is facilitated by using dialdehyde, carbodiimidecoupling, homobifunctional crosslinkers and heterobifunctionalcrosslinkers as described above for coupling an antibody with a memberof a specific binding pair.

The labeling method chosen depends on the functional groups available onthe enzyme and the material to be labeled, and the tolerance of both tothe conjugation conditions. The labeling method used in the presentinvention can be one of, but not limited to, any conventional methodscurrently employed including those described by Engvall and Pearlmann,Immunochemistry 8, 871 (1971), Avrameas and Ternynck, Immunochemistry 8,1175 (1975), Ishikawa et al., J. Immunoassay 4(3):209-327 (1983) andJablonski, Anal. Biochem. 148:199 (1985).

Labeling can be accomplished by indirect methods such as using spacersor other members of specific binding pairs. An example of this is thedetection of a biotinylated antibody with unlabeled streptavidin andbiotinylated enzyme, with streptavidin and biotinylated enzyme beingadded either sequentially or simultaneously. Thus, according to thepresent invention, the antibody used to detect can be detectably-labeleddirectly with a reporter or indirectly with a first member of a specificbinding pair. When the antibody is coupled to a first member of aspecific binding pair, then detection is effected by reacting theantibody-first member of a specific binding complex with the secondmember of the binding pair that is labeled or unlabeled as mentionedabove.

Moreover, the unlabeled detector antibody can be detected by reactingthe unlabeled antibody with a labeled antibody specific for theunlabeled antibody. In this instance “detectably-labeled” as used aboveis taken to mean containing an epitope by which an antibody specific forthe unlabeled antibody can bind. Such an anti-antibody can be labeleddirectly or indirectly using any of the approaches discussed above. Forexample, the anti-antibody can be coupled to biotin which is detected byreacting with the streptavidin-horseradish peroxidase system discussedabove.

In one embodiment of this invention biotin is utilized. The biotinylatedantibody is in turn reacted with streptavidin-horseradish peroxidasecomplex. Orthophenylenediamine, 4-chloro-naphthol, tetramethylbenzidine(TMB), ABTS, BTS or ASA can be used to effect chromogenic detection.

In one immunoassay format for practicing this invention, a forwardsandwich assay is used in which the capture reagent has beenimmobilized, using conventional techniques, on the surface of a support.Suitable supports used in assays include synthetic polymer supports,such as polypropylene, polystyrene, substituted polystyrene, e.g.aminated or carboxylated polystyrene, polyacrylamides, polyamides,polyvinylchloride, glass beads, agarose, or nitrocellulose.

Agents, or compositions comprising such agents, that inhibit tumor cellsfrom undergoing an epithelial to mesenchymal transition, inhibit tumorcells that have undergone an epithelial to mesenchymal transition, orstimulate mesenchymal-like tumor cells to undergo a mesenchymal toepithelial transition, identified by the methods described herein, canbe used in methods for treating tumors or tumor metastases in a patient.

As used herein, the term “patient” preferably refers to a human in needof treatment with an anti-cancer agent for any purpose, and morepreferably a human in need of such a treatment to treat cancer, or aprecancerous condition or lesion. However, the term “patient” can alsorefer to non-human animals, preferably mammals such as dogs, cats,horses, cows, pigs, sheep and non-human primates, among others, that arein need of treatment with an anti-cancer agent.

Anti-cancer agents identified by any of the methods described herein (orcompositions comprising them) can be used to treat any of the followingtumors or cancers: NSCL, breast, colon, or pancreatic cancer, lungcancer, bronchioloalveolar cell lung cancer, bone cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, gastric cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the vagina,carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, prostate cancer, cancer of the bladder, cancer of the ureter,cancer of the kidney, renal cell carcinoma, carcinoma of the renalpelvis, mesothelioma, hepatocellular cancer, biliary cancer, chronic oracute leukemia, lymphocytic lymphomas, neoplasms of the central nervoussystem (CNS), spinal axis tumors, brain stem glioma, glioblastomamultiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas,meningiomas, squamous cell carcinomas, pituitary adenomas, includingrefractory versions of any of the above cancers, or a combination of oneor more of the above cancers.

The term “refractory” as used herein is used to define a cancer forwhich treatment (e.g. chemotherapy drugs, biological agents, and/orradiation therapy) has proven to be ineffective. A refractory cancertumor may shrink, but not to the point where the treatment is determinedto be effective. Typically however, the tumor stays the same size as itwas before treatment (stable disease), or it grows (progressivedisease).

Anti-cancer agents identified by any of the methods described herein (orcompositions comprising them) can be used to treat abnormal cell growth.

It will be appreciated by one of skill in the medical arts that theexact manner of administering to said patient of a therapeuticallyeffective amount of the identified agent, e.g. in a combination of anEGFR kinase inhibitor and said agent, will be at the discretion of theattending physician. The mode of administration, including dosage,combination with other anti-cancer agents, timing and frequency ofadministration, and the like, may be affected by e.g. a diagnosis of apatient's likely responsiveness to an EGFR or IGFR kinase inhibitor, aswell as the patient's condition and history. Thus, even patientsdiagnosed with tumors predicted to be relatively sensitive to e.g. anEGFR or IGFR kinase inhibitor as a single agent may still benefit fromtreatment with a combination of such a kinase inhibitor and theidentified agent, optionally in combination with other anti-canceragents, or other agents that may alter a tumor's sensitivity to kinaseinhibitors.

In the context of this invention, an “effective amount” of an agent ortherapy is as defined above. A “sub-therapeutic amount” of an agent ortherapy is an amount less than the effective amount for that agent ortherapy, but when combined with an effective or sub-therapeutic amountof another agent or therapy can produce a result desired by thephysician, due to, for example, synergy in the resulting efficaciouseffects, or reduced side effects.

Additionally, the present invention provides a pharmaceuticalcomposition comprising a combination of for example an EGFR or IGFRkinase inhibitor and the identified agent in a pharmaceuticallyacceptable carrier.

The invention also encompasses a pharmaceutical composition prepared byany of the methods described herein, that is comprised of an agent thatinhibits tumor cells from undergoing an epithelial to mesenchymaltransition, inhibit tumor cells that have undergone an epithelial tomesenchymal transition, or stimulate mesenchymal-like tumor cells toundergo a mesenchymal to epithelial transition, identified by any of themethods described herein (i.e. “the identified agent”), in combinationwith a pharmaceutically acceptable carrier, and optionally incombination with one or more other anti-cancer agents (e.g. an EGFR,IGF-1R, RON or MET receptor tyrosine kinase inhibitor).

Methods of preparing pharmaceutical compositions are well known in theart, as for example described in references such as Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18^(th)edition (1990).

Preferably the composition is comprised of a pharmaceutically acceptablecarrier and a non-toxic therapeutically effective amount of theidentified agent (including pharmaceutically acceptable salts of eachcomponent thereof).

Moreover, within this preferred embodiment, the invention encompasses apharmaceutical composition for the treatment of disease, the use ofwhich results in the inhibition of growth of neoplastic cells, benign ormalignant tumors, or metastases, comprising a pharmaceuticallyacceptable carrier and a non-toxic therapeutically effective amount ofthe identified agent (including pharmaceutically acceptable salts ofeach component thereof).

The term “pharmaceutically acceptable salts” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids. When acompound of the present invention is acidic, its corresponding salt canbe conveniently prepared from pharmaceutically acceptable non-toxicbases, including inorganic bases and organic bases. Salts derived fromsuch inorganic bases include aluminum, ammonium, calcium, copper (cupricand cuprous), ferric, ferrous, lithium, magnesium, manganese (manganicand manganous), potassium, sodium, zinc and the like salts. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines, as wellas cyclic amines and substituted amines such as naturally occurring andsynthesized substituted amines. Other pharmaceutically acceptableorganic non-toxic bases from which salts can be formed include ionexchange resins such as, for example, arginine, betaine, caffeine,choline, N′,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylameine, trimethylamine,tripropylamine, tromethamine and the like.

When a compound of the present invention is basic, its correspondingsalt can be conveniently prepared from pharmaceutically acceptablenon-toxic acids, including inorganic and organic acids. Such acidsinclude, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic,citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like.Particularly preferred are citric, hydrobromic, hydrochloric, maleic,phosphoric, sulfuric and tartaric acids.

The pharmaceutical compositions of the present invention comprise theidentified agent (including pharmaceutically acceptable salts thereof)as active ingredient, a pharmaceutically acceptable carrier andoptionally other therapeutic ingredients or adjuvants. Other therapeuticagents may include those cytotoxic, chemotherapeutic or anti-canceragents, or agents which enhance the effects of such agents, as listedabove. The compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions may be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

In practice, the compounds represented by the identified agent(including pharmaceutically acceptable salts thereof) of this inventioncan be combined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.oral or parenteral (including intravenous). Thus, the pharmaceuticalcompositions of the present invention can be presented as discrete unitssuitable for oral administration such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient.Further, the compositions can be presented as a powder, as granules, asa solution, as a suspension in an aqueous liquid, as a non-aqueousliquid, as an oil-in-water emulsion, or as a water-in-oil liquidemulsion. In addition to the common dosage forms set out above, theidentified agent (including pharmaceutically acceptable salts thereof)may also be administered by controlled release means and/or deliverydevices. The combination compositions may be prepared by any of themethods of pharmacy. In general, such methods include a step of bringinginto association the active ingredients with the carrier thatconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both. The product can then be conveniently shaped into the desiredpresentation.

Thus, the pharmaceutical compositions of this invention may include apharmaceutically acceptable carrier and a combination of the identifiedagent (including pharmaceutically acceptable salts thereof). Theidentified agent (including pharmaceutically acceptable salts thereof),can also be included in pharmaceutical compositions in combination withone or more other therapeutically active compounds. Othertherapeutically active compounds may include those cytotoxic,chemotherapeutic or anti-cancer agents, or agents which enhance theeffects of such agents, as listed above.

Thus in one embodiment of this invention, a pharmaceutical compositioncan comprise the identified agent in combination with an anticanceragent, wherein said anti-cancer agent is a member selected from thegroup consisting of alkylating drugs, antimetabolites, microtubuleinhibitors, podophyllotoxins, antibiotics, nitrosoureas, hormonetherapies, kinase inhibitors, activators of tumor cell apoptosis, andantiangiogenic agents.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media may be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents, and the likemay be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like may be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets may be coated by standard aqueous or nonaqueoustechniques.

A tablet containing the composition of this invention may be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent. Eachtablet preferably contains from about 0.05 mg to about 5 g of the activeingredient and each cachet or capsule preferably contains from about0.05 mg to about 5 g of the active ingredient.

For example, a formulation intended for the oral administration tohumans may contain from about 0.5 mg to about 5 g of active agent,compounded with an appropriate and convenient amount of carrier materialthat may vary from about 5 to about 95 percent of the total composition.Unit dosage forms will generally contain between from about 1 mg toabout 2 g of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

Pharmaceutical compositions of the present invention suitable forparenteral administration may be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical sue such as, for example, an aerosol, cream,ointment, lotion, dusting powder, or the like. Further, the compositionscan be in a form suitable for use in transdermal devices. Theseformulations may be prepared, utilizing the identified agent (includingpharmaceutically acceptable salts thereof) of this invention, viaconventional processing methods. As an example, a cream or ointment isprepared by admixing hydrophilic material and water, together with about5 wt % to about 10 wt % of the compound, to produce a cream or ointmenthaving a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories may be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in molds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above may include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining the identified agent (including pharmaceutically acceptablesalts thereof) may also be prepared in powder or liquid concentrateform.

In the context of this invention, other anticancer agents includes, forexample, other cytotoxic, chemotherapeutic or anti-cancer agents, orcompounds that enhance the effects of such agents, anti-hormonal agents,angiogenesis inhibitors, tumor cell pro-apoptotic orapoptosis-stimulating agents, signal transduction inhibitors,anti-proliferative agents, anti-HER2 antibody or animmunotherapeutically active fragment thereof, anti-proliferativeagents, COX II (cyclooxygenase II) inhibitors, and agents capable ofenhancing antitumor immune responses.

In the context of this invention, other cytotoxic, chemotherapeutic oranti-cancer agents, or compounds that enhance the effects of suchagents, include, for example: alkylating agents or agents with analkylating action, such as cyclophosphamide (CTX; e.g. CYTOXAN®),chlorambucil (CHL; e.g. LEUKERAN®), cisplatin (C is P; e.g. PLATINOL®)busulfan (e.g. MYLERAN®), melphalan, carmustine (BCNU), streptozotocin,triethylenemelamine (TEM), mitomycin C, and the like; anti-metabolites,such as methotrexate (MTX), etoposide (VP16; e.g. VEPESID®),6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C),5-fluorouracil (5-FU), capecitabine (e.g. XELODA®), dacarbazine (DTIC),and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g.ADRIAMYCIN®), daunorubicin (daunomycin), bleomycin, mithramycin and thelike; alkaloids, such as vinca alkaloids such as vincristine (VCR),vinblastine, and the like; and other antitumor agents, such aspaclitaxel (e.g. TAXOL®) and pactitaxel derivatives, the cytostaticagents, glucocorticoids such as dexamethasone (DEX; e.g. DECADRON®) andcorticosteroids such as prednisone, nucleoside enzyme inhibitors such ashydroxyurea, amino acid depleting enzymes such as asparaginase,leucovorin and other folic acid derivatives, and similar, diverseantitumor agents. The following agents may also be used as additionalagents: arnifostine (e.g. ETHYOL®), dactinomycin, mechlorethamine(nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU),doxorubicin lipo (e.g. DOXIL®), gemcitabine (e.g. GEMZAR®), daunorubicinlipo (e.g. DAUNOXOMEC), procarbazine, mitomycin, docetaxel (e.g.TAXOTERE®), aldesleukin, carboplatin, oxaliplatin, cladribine,camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin(SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna,interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide,megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen,teniposide, testolactone, thioguanine, thiotepa, uracil mustard,vinorelbine, chlorambucil.

As used herein, the term “anti-hormonal agent” includes natural orsynthetic organic or peptidic compounds that act to regulate or inhibithormone action on tumors. Antihormonal agents include, for example:steroid receptor antagonists, anti-estrogens such as tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, other aromataseinhibitors, 42-hydroxytamoxifen, trioxifene, keoxifene, LY 117018,onapristone, and toremifene (e.g. FARESTON®); anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; agonists and/or antagonists of glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH) and LHRH (leuteinizing hormone-releasinghormone); the LHRH agonist goserelin acetate, commercially available asZOLADEX® (AstraZeneca); the LHRH antagonist D-alaninamideN-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-N-6-(3-pyridinylcarbonyl)-L-lysyl-N-6-(3-pyridinylcarbonyl)-D-lysyl-L-leucyl-N-6-(1-methylethyl)-L-lysyl-L-proline(e.g. ANTIDE®, Ares-Serono); the LHRH antagonist ganirelix acetate; thesteroidal anti-androgens cyproterone acetate (CPA) and megestrolacetate, commercially available as MEGACE® (Bristol-Myers Oncology); thenonsteroidal anti-androgen flutamide(2-methyl-N-[4,20-nitro-3-(trifluoromethyl) phenylpropanamide),commercially available as EULEXIN® (Schering Corp.); the non-steroidalanti-androgen nilutamide,(5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4′-nitrophenyl)-4,4-dimethyl-imidazolidine-dione);and antagonists for other non-permissive receptors, such as antagonistsfor RAR, RXR, TR, VDR, and the like.

Anti-angiogenic agents include, for example: VEGFR inhibitors, such asSU-5416 and SU-6668 (Sugen Inc. of South San Francisco, Calif., USA), oras described in, for example International Application Nos. WO 99/24440,WO 99/62890, WO 95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO98/02437, and U.S. Pat. Nos. 5,883,113, 5,886,020, 5,792,783, 5,834,504and 6,235,764; VEGF inhibitors such as IM862 (Cytran Inc. of Kirkland,Wash., USA); angiozyme, a synthetic ribozyme from Ribozyme (Boulder,Colo.) and Chiron (Emeryville, Calif.); and antibodies to VEGF, such asbevacizumab (e.g. AVASTIN™, Genentech, South San Francisco, Calif.), arecombinant humanized antibody to VEGF; integrin receptor antagonistsand integrin antagonists, such as to α_(v)β₃, α_(v)β₅ and α_(v)β₆integrins, and subtypes thereof, e.g. cilengitide (EMD 121974), or theanti-integrin antibodies, such as for example α_(v)β₃ specific humanizedantibodies (e.g. VITAXIN®); factors such as IFN-alpha (U.S. Pat. Nos.41,530,901, 4,503,035, and 5,231,176); angiostatin and plasminogenfragments (e.g. kringle 1-4, kringle 5, kringle 1-3 (O'Reilly, M. S. etal. (1994) Cell 79:315-328; Cao et al. (1996) J. Biol. Chem. 271:29461-29467; Cao et al. (1997) J. Biol. Chem. 272:22924-22928);endostatin (O'Reilly, M. S. et al. (1997) Cell 88:277; and InternationalPatent Publication No. WO 97/15666); thrombospondin (TSP-1; Frazier,(1991) Curr. Opin. Cell Biol. 3:792); platelet factor 4 (PF4);plasminogen activator/urokinase inhibitors; urokinase receptorantagonists; heparinases; fumagillin analogs such as TNP-4701; suraminand suramin analogs; angiostatic steroids; bFGF antagonists; flk-1 andflt-1 antagonists; anti-angiogenesis agents such as MMP-2(matrix-metalloproteinase 2) inhibitors and MMP-9(matrix-metalloproteinase 9) inhibitors. Examples of useful matrixmetalloproteinase inhibitors are described in International PatentPublication Nos. WO 96/33172, WO 96/27583, WO 98/07697, WO 98/03516, WO98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90/05719, WO99/52910, WO 99/52889, WO 99/29667, and WO 99/07675, European PatentPublication Nos. 818,442, 780,386, 1,004,578, 606,046, and 931,788;Great Britain Patent Publication No. 9912961, and U.S. Pat. Nos.5,863,949 and 5,861,510. Preferred MMP-2 and MMP-9 inhibitors are thosethat have little or no activity inhibiting MMP-1. More preferred, arethose that selectively inhibit MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

Signal transduction inhibitors include, for example: erbB2 receptorinhibitors, such as organic molecules, or antibodies that bind to theerbB2 receptor, for example, trastuzumab (e.g. HERCEPTIN®); inhibitorsof other protein tyrosine-kinases, e.g. imitinib (e.g. GLEEVEC®); rasinhibitors; raf inhibitors; MEK inhibitors; mTOR inhibitors; cyclindependent kinase inhibitors; protein kinase C inhibitors; and PDK-1inhibitors (see Dancey, J. and Sausville, E. A. (2003) Nature Rev. DrugDiscovery 2:92-313, for a description of several examples of suchinhibitors, and their use in clinical trials for the treatment ofcancer).

ErbB2 receptor inhibitors include, for example: ErbB2 receptorinhibitors, such as GW-282974 (Glaxo Wellcome plc), monoclonalantibodies such as AR-209 (Aronex Pharmaceuticals Inc. of The Woodlands,Tex., USA) and 2B-1 (Chiron), and erbB2 inhibitors such as thosedescribed in International Publication Nos. WO 98/02434, WO 99/35146, WO99/35132, WO 98/02437, WO 97/13760, and WO 95/19970, and U.S. Pat. Nos.5,587,458, 5,877,305, 6,465,449 and 6,541,481.

Antiproliferative agents include, for example: Inhibitors of the enzymefarnesyl protein transferase and inhibitors of the receptor tyrosinekinase PDGFR, including the compounds disclosed and claimed in U.S. Pat.Nos. 6,080,769, 6,194,438, 6,258,824, 6,586,447, 6,071,935, 6,495,564,6,150,377, 6,596,735 and 6,479,513, and International Patent PublicationWO 01/40217. Antiproliferative agents also include inhibitors of thereceptor tyrosine kinases IGF-1R and FGFR.

Examples of useful COX-II inhibitors include alecoxib (e.g. CELEBREX™),valdecoxib, and rofecoxib. Agents capable of enhancing antitumor immuneresponses include, for example: CTLA4 (cytotoxic lymphocyte antigen 4)antibodies (e.g. MDX-CTLA4), and other agents capable of blocking CTLA4.Specific CTLA4 antibodies that can be used in the present inventioninclude those described in U.S. Pat. No. 6,682,736.

The present invention further provides a method for treating tumors ortumor metastases in a patient, comprising administering to the patientsimultaneously or sequentially a therapeutically effective amount of theidentified agent described herein above and optionally one or more otheranticancer agents.

Dosage levels for the identified agents of this invention will be asdescribed herein, but will depend upon a variety of factors includingthe age, body weight, general health, sex, diet, time of administration,route of administration, rate of excretion, drug combination and theseverity of the particular disease undergoing therapy.

The use of the cytotoxic and other anticancer agents described above inchemotherapeutic regimens is generally well characterized in the cancertherapy arts, and their use herein falls under the same considerationsfor monitoring tolerance and effectiveness and for controllingadministration routes and dosages, with some adjustments. For example,the actual dosages of the cytotoxic agents may vary depending upon thepatient's cultured cell response determined by using histoculturemethods. Generally, the dosage will be reduced compared to the amountused in the absence of additional other agents.

Typical dosages of an effective cytotoxic agent can be in the rangesrecommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

As used herein, the term “EGFR kinase inhibitor” refers to any EGFRkinase inhibitor that is currently known in the art or that will beidentified in the future, and includes any chemical entity that, uponadministration to a patient, results in inhibition of a biologicalactivity associated with activation of the EGF receptor in the patient,including any of the downstream biological effects otherwise resultingfrom the binding to EGFR of its natural ligand. Such EGFR kinaseinhibitors include any agent that can block EGFR activation or any ofthe downstream biological effects of EGFR activation that are relevantto treating cancer in a patient. Such an inhibitor can act by bindingdirectly to the intracellular domain of the receptor and inhibiting itskinase activity. Alternatively, such an inhibitor can act by occupyingthe ligand binding site or a portion thereof of the EGF receptor,thereby making the receptor inaccessible to its natural ligand so thatits normal biological activity is prevented or reduced. Alternatively,such an inhibitor can act by modulating the dimerization of EGFRpolypeptides, or interaction of EGFR polypeptide with other proteins, orenhance ubiquitination and endocytotic degradation of EGFR. EGFR kinaseinhibitors include but are not limited to low molecular weightinhibitors, antibodies or antibody fragments, peptide or RNA aptamers,antisense constructs, small inhibitory RNAs (i.e. RNA interference bydsRNA; RNAi), and ribozymes. In a preferred embodiment, the EGFR kinaseinhibitor is a small organic molecule or an antibody that bindsspecifically to the human EGFR.

EGFR kinase inhibitors include, for example quinazoline EGFR kinaseinhibitors, pyrido-pyrimidine EGFR kinase inhibitors,pyrimido-pyrimidine EGFR kinase inhibitors, pyrrolo-pyrimidine EGFRkinase inhibitors, pyrazolo-pyrimidine EGFR kinase inhibitors,phenylamino-pyrimidine EGFR kinase inhibitors, oxindole EGFR kinaseinhibitors, indolocarbazole EGFR kinase inhibitors, phthalazine EGFRkinase inhibitors, isoflavone EGFR kinase inhibitors, quinalone EGFRkinase inhibitors, and tyrphostin EGFR kinase inhibitors, such as thosedescribed in the following patent publications, and all pharmaceuticallyacceptable salts and solvates of said EGFR kinase inhibitors:International Patent Publication Nos. WO 96/33980, WO 96/30347, WO97/30034, WO 97/30044, WO 97/38994, WO 97/49688, WO 98/02434, WO97/38983, WO 95/19774, WO 95/19970, WO 97/13771, WO 98/02437, WO98/02438, WO 97/32881, WO 98/33798, WO 97/32880, WO 97/3288, WO97/02266, WO 97/27199, WO 98/07726, WO 97/34895, WO 96/31510, WO98/14449, WO 98/14450, WO 98/14451, WO 95/09847, WO 97/19065, WO98/17662, WO 99/35146, WO 99/35132, WO 99/07701, and WO 92/20642;European Patent Application Nos. EP 520722, EP 566226, EP 787772, EP837063, and EP 682027; U.S. Pat. Nos. 5,747,498, 5,789,427, 5,650,415,and 5,656,643; and German Patent Application No. DE 19629652. Additionalnon-limiting examples of low molecular weight EGFR kinase inhibitorsinclude any of the EGFR kinase inhibitors described in Traxler, P.,1998, Exp. Opin. Ther. Patents 8(12):1599-1625.

Specific preferred examples of low molecular weight EGFR kinaseinhibitors that can be used according to the present invention include[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl) amine(also known as OSI-774, erlotinib, or TARCEVA® (erlotinib HCl); OSIPharmaceuticals/Genentech/Roche) (U.S. Pat. No. 5,747,498; InternationalPatent Publication No. WO 01/34574, and Moyer, J. D. et al. (1997)Cancer Res. 57:4838-4848); CI-1033 (formerly known as PD183805; Pfizer)(Sherwood et al., 1999, Proc. Am. Assoc. Cancer Res. 40:723); PD-158780(Pfizer); AG-1478 (University of California); CGP-59326 (Novartis);PKI-166 (Novartis); EKB-569 (Wyeth); GW-2016 (also known as GW-572016 orlapatinib ditosylate; GSK); and gefitinib (also known as ZD1839 orIRESSA™; Astrazeneca) (Woodburn et al., 1997, Proc. Am. Assoc. CancerRes. 38:633). A particularly preferred low molecular weight EGFR kinaseinhibitor that can be used according to the present invention is[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl) amine(i.e. erlotinib), its hydrochloride salt (i.e. erlotinib HCl, TARCEVA®),or other salt forms (e.g. erlotinib mesylate).

EGFR kinase inhibitors also include, for example multi-kinase inhibitorsthat have activity on EGFR kinase, i.e. inhibitors that inhibit EGFRkinase and one or more additional kinases. Examples of such compoundsinclude the EGFR and HER2 inhibitor CI-1033 (formerly known as PD183805;Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW-572016 orlapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (atyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma);BIBW-2992, an irreversible dual EGFR/HER2 kinase inhibitor (BoehringerIngelheim Corp.); the EGFR and HER2 inhibitor EKB-569 (Wyeth); theVEGF-R2 and EGFR inhibitor ZD6474 (also known as ZACTIMA™; AstraZenecaPharmaceuticals), and the EGFR and HER2 inhibitor BMS-599626(Bristol-Myers Squibb).

Antibody-based EGFR kinase inhibitors include any anti-EGFR antibody orantibody fragment that can partially or completely block EGFR activationby its natural ligand. Non-limiting examples of antibody-based EGFRkinase inhibitors include those described in Modjtahedi, H., et al.,1993, Br. J. Cancer 67:247-253; Teramoto, T., et al., 1996, Cancer77:639-645; Goldstein et al., 1995, Clin. Cancer Res. 1:1311-1318;Huang, S. M., et al., 1999, Cancer Res. 15:59(8):1935-40; and Yang, X.,et al., 1999, Cancer Res. 59:1236-1243. Thus, the EGFR kinase inhibitorcan be the monoclonal antibody Mab E7.6.3 (Yang, X. D. et al. (1999)Cancer Res. 59:1236-43), or Mab C225 (ATCC Accession No. HB-8508), or anantibody or antibody fragment having the binding specificity thereof.Suitable monoclonal antibody EGFR kinase inhibitors include, but are notlimited to, IMC-C225 (also known as cetuximab or ERBITUX™; ImcloneSystems), ABX-EGF (Abgenix), EMD 72000 (Merck KgaA, Darmstadt), RH3(York Medical Bioscience Inc.), and MDX-447 (Medarex/Merck KgaA).

EGFR kinase inhibitors for use in the present invention canalternatively be peptide or RNA aptamers. Such aptamers can for exampleinteract with the extracellular or intracellular domains of EGFR toinhibit EGFR kinase activity in cells. An aptamer that interacts withthe extracellular domain is preferred as it would not be necessary forsuch an aptamer to cross the plasma membrane of the target cell. Anaptamer could also interact with the ligand for EGFR (e.g. EGF, TGF-α),such that its ability to activate EGFR is inhibited. Methods forselecting an appropriate aptamer are well known in the art. Such methodshave been used to select both peptide and RNA aptamers that interactwith and inhibit EGFR family members (e.g. see Buerger, C. et al. et al.(2003) J. Biol. Chem. 278:37610-37621; Chen, C-H. B. et al. (2003) Proc.Natl. Acad. Sci. 100:9226-9231; Buerger, C. and Groner, B. (2003) J.Cancer Res. Clin. Oncol. 129(12):669-675. Epub 2003 Sep. 11.).

EGFR kinase inhibitors for use in the present invention canalternatively be based on antisense oligonucleotide constructs.Anti-sense oligonucleotides, including anti-sense RNA molecules andanti-sense DNA molecules, would act to directly block the translation ofEGFR mRNA by binding thereto and thus preventing protein translation orincreasing mRNA degradation, thus decreasing the level of EGFR kinaseprotein, and thus activity, in a cell. For example, antisenseoligonucleotides of at least about 15 bases and complementary to uniqueregions of the mRNA transcript sequence encoding EGFR can besynthesized, e.g., by conventional phosphodiester techniques andadministered by e.g., intravenous injection or infusion. Methods forusing antisense techniques for specifically inhibiting gene expressionof genes whose sequence is known are well known in the art (e.g. seeU.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091;6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as EGFR kinaseinhibitors for use in the present invention. EGFR gene expression can bereduced by contacting the tumor, subject or cell with a small doublestranded RNA (dsRNA), or a vector or construct causing the production ofa small double stranded RNA, such that expression of EGFR isspecifically inhibited (i.e. RNA interference or RNAi). Methods forselecting an appropriate dsRNA or dsRNA-encoding vector are well knownin the art for genes whose sequence is known (e.g. see Tuschi, T., etal. (1999) Genes Dev. 13(24):3191-3197; Elbashir, S. M. et al. (2001)Nature 411:494-498; Hannon, G. J. (2002) Nature 418:244-251; McManus, M.T. and Sharp, P. A. (2002) Nature Reviews Genetics 3:737-747;Bremmelkamp, T. R. et al. (2002) Science 296:550-553; U.S. Pat. Nos.6,573,099 and 6,506,559; and International Patent Publication Nos. WO01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as EGFR kinase inhibitors for use in thepresent invention. Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage of RNA. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by endonucleolytic cleavage.Engineered hairpin or hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of EGFRmRNA sequences are thereby useful within the scope of the presentinvention. Specific ribozyme cleavage sites within any potential RNAtarget are initially identified by scanning the target molecule forribozyme cleavage sites, which typically include the followingsequences, GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween about 15 and 20 ribonucleotides corresponding to the region ofthe target gene containing the cleavage site can be evaluated forpredicted structural features, such as secondary structure, that canrender the oligonucleotide sequence unsuitable. The suitability ofcandidate targets can also be evaluated by testing their accessibilityto hybridization with complementary oligonucleotides, using, e.g.,ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as EGFR kinaseinhibitors can be prepared by known methods. These include techniquesfor chemical synthesis such as, e.g., by solid phase phosphoramaditechemical synthesis. Alternatively, anti-sense RNA molecules can begenerated by in vitro or in vivo transcription of DNA sequences encodingthe RNA molecule. Such DNA sequences can be incorporated into a widevariety of vectors that incorporate suitable RNA polymerase promoterssuch as the T7 or SP6 polymerase promoters. Various modifications to theoligonucleotides of the invention can be introduced as a means ofincreasing intracellular stability and half-life. Possible modificationsinclude but are not limited to the addition of flanking sequences ofribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′-O-methyl rather thanphosphodiesterase linkages within the oligonucleotide backbone.

As used herein, the term “IGF-1R kinase inhibitor” refers to any IGF-1Rkinase inhibitor that is currently known in the art or that will beidentified in the future, and includes any chemical entity that, uponadministration to a patient, results in inhibition of a biologicalactivity associated with activation of the IGF-1 receptor in thepatient, including any of the downstream biological effects otherwiseresulting from the binding to IGF-1R of its natural ligand. Such IGF-1Rkinase inhibitors include any agent that can block IGF-1R activation orany of the downstream biological effects of IGF-1R activation that arerelevant to treating cancer in a patient. Such an inhibitor can act bybinding directly to the intracellular domain of the receptor andinhibiting its kinase activity. Alternatively, such an inhibitor can actby occupying the ligand binding site or a portion thereof of the IGF-1receptor, thereby making the receptor inaccessible to its natural ligandso that its normal biological activity is prevented or reduced.Alternatively, such an inhibitor can act by modulating the dimerizationof IGF-1R polypeptides, or interaction of IGF-1R polypeptide with otherproteins, or enhance ubiquitination and endocytotic degradation ofIGF-1R. An IGF-1R kinase inhibitor can also act by reducing the amountof IGF-1 available to activate IGF-1R, by for example antagonizing thebinding of IGF-1 to its receptor, by reducing the level of IGF-1, or bypromoting the association of IGF-1 with proteins other than IGF-1R suchas IGF binding proteins (e.g. IGFBP3). IGF-1R kinase inhibitors includebut are not limited to low molecular weight inhibitors, antibodies orantibody fragments, antisense constructs, small inhibitory RNAs (i.e.RNA interference by dsRNA; RNAi), and ribozymes. In a preferredembodiment, the IGF-1R kinase inhibitor is a small organic molecule oran antibody that binds specifically to the human IGF-1R.

IGF-1R kinase inhibitors include, for example imidazopyrazine IGF-1Rkinase inhibitors, azabicyclic amine inhibitors, quinazoline IGF-1Rkinase inhibitors, pyrido-pyrimidine IGF-1R kinase inhibitors,pyrimido-pyrimidine IGF-1R kinase inhibitors, pyrrolo-pyrimidine IGF-1Rkinase inhibitors, pyrazolo-pyrimidine IGF-1R kinase inhibitors,phenylamino-pyrimidine IGF-1R kinase inhibitors, oxindole IGF-1R kinaseinhibitors, indolocarbazole IGF-1R kinase inhibitors, phthalazine IGF-1Rkinase inhibitors, isoflavone IGF-1R kinase inhibitors, quinalone IGF-1Rkinase inhibitors, and tyrphostin IGF-1R kinase inhibitors, and allpharmaceutically acceptable salts and solvates of such IGF-1R kinaseinhibitors.

Examples of IGF-1R kinase inhibitors include those in InternationalPatent Publication No. WO 05/097800, that describes azabicyclic aminederivatives, International Patent Publication No. WO 05/037836, thatdescribes imidazopyrazine IGF-1R kinase inhibitors, International PatentPublication Nos. WO 03/018021 and WO 03/018022, that describepyrimidines for treating IGF-1R related disorders, International PatentPublication Nos. WO 02/102804 and WO 02/102805, that describecyclolignans and cyclolignans as IGF-1R inhibitors, International PatentPublication No. WO 02/092599, that describes pyrrolopyrimidines for thetreatment of a disease which responds to an inhibition of the IGF-1Rtyrosine kinase, International Patent Publication No. WO 01/72751, thatdescribes pyrrolopyrimidines as tyrosine kinase inhibitors, and inInternational Patent Publication No. WO 00/71129, that describespyrrolotriazine inhibitors of kinases, and in International PatentPublication No. WO 97/28161, that describes pyrrolo[2,3-d]pyrimidinesand their use as tyrosine kinase inhibitors, Parrizas, et al., whichdescribes tyrphostins with in vitro and in vivo IGF-1R inhibitoryactivity (Endocrinology, 138:1427-1433 (1997)), International PatentPublication No. WO 00/35455, that describes heteroaryl-aryl ureas asIGF-1R inhibitors, International Patent Publication No. WO 03/048133,that describes pyrimidine derivatives as modulators of IGF-1R,International Patent Publication No. WO 03/024967, WO 03/035614, WO03/035615, WO 03/035616, and WO 03/035619, that describe chemicalcompounds with inhibitory effects towards kinase proteins, InternationalPatent Publication No. WO 03/068265, that describes methods andcompositions for treating hyperproliferative conditions, InternationalPatent Publication No. WO 00/17203, that describes pyrrolopyrimidines asprotein kinase inhibitors, Japanese Patent Publication No. JP07/133,280, that describes a cephem compound, its production andantimicrobial composition, Albert, A. et al., Journal of the ChemicalSociety, 11: 1540-1547 (1970), which describes pteridine studies andpteridines unsubstituted in the 4-position, and A. Albert et al., Chem.Biol. Pteridines Proc. Int. Symp., 4th, 4: 1-5 (1969) which describes asynthesis of pteridines (unsubstituted in the 4-position) frompyrazines, via 3-4-dihydropteridines.

Additional, specific examples of IGF-1R kinase inhibitors that can beused according to the present invention include h7C10 (Centre deRecherche Pierre Fabre), an IGF-1 antagonist; EM-164 (ImmunoGen Inc.),an IGF-1R modulator; CP-751871 (Pfizer Inc.), an IGF-1 antagonist;lanreotide (Ipsen), an IGF-1 antagonist; IGF-1R oligonucleotides (LynxTherapeutics Inc.); IGF-1 oligonucleotides (National Cancer Institute);IGF-1R protein-tyrosine kinase inhibitors in development by Novartis(e.g. NVP-AEW541, Garcia-Echeverria, C. et al. (2004) Cancer Cell5:231-239; or NVP-ADW742, Mitsiades, C. S. et al. (2004) Cancer Cell5:221-230); IGF-1R protein-tyrosine kinase inhibitors (Ontogen Corp);OSI-906 (OSI Pharmaceuticals); AG-1024 (Camirand, A. et al. (2005)Breast Cancer Research 7:R570-R579 (DOI 10.1186/bcr1028); Camirand, A.and Pollak, M. (2004) Brit. J. Cancer 90:1825-1829; Pfizer Inc.), anIGF-1 antagonist; the tyrphostins AG-538 and I-OMe-AG 538; BMS-536924, asmall molecule inhibitor of IGF-1R; PNU-145156E (Pharmacia & UpjohnSpA), an IGF-1 antagonist; BMS 536924, a dual IGF-1R and IR kinaseinhibitor (Bristol-Myers Squibb); AEW541 (Novartis); GSK621659A (GlaxoSmith-Kline); INSM-18 (Insmed); and XL-228 (Exelixis).

Antibody-based IGF-1R kinase inhibitors include any anti-IGF-1R antibodyor antibody fragment that can partially or completely block IGF-1Ractivation by its natural ligand. Antibody-based IGF-1R kinaseinhibitors also include any anti-IGF-1 antibody or antibody fragmentthat can partially or completely block IGF-1R activation. Non-limitingexamples of antibody-based IGF-1R kinase inhibitors include thosedescribed in Larsson, O. et al (2005) Brit. J. Cancer 92:2097-2101 andIbrahim, Y. H. and Yee, D. (2005) Clin. Cancer Res. 11:944s-950s; orbeing developed by Imclone (e.g. IMC-A12), or AMG-479, an anti-IGF-1Rantibody (Amgen); R1507, an anti-IGF-1R antibody (Genmab/Roche);AVE-1642, an anti-IGF-1R antibody (Immunogen/Sanofi-Aventis); MK 0646 orh7C10, an anti-IGF-1R antibody (Merck); or antibodies being develop bySchering-Plough Research Institute (e.g. SCH 717454 or 19D12; or asdescribed in US Patent Application Publication Nos. US 2005/0136063 A1and US 2004/0018191 A1). The IGF-1R kinase inhibitor can be a monoclonalantibody, or an antibody or antibody fragment having the bindingspecificity thereof.

As used herein, the term “PDGFR kinase inhibitor” refers to any PDGFRkinase inhibitor that is currently known in the art or that will beidentified in the future, and includes any chemical entity that, uponadministration to a patient, results in inhibition of a biologicalactivity associated with activation of the PDGF receptor in the patient,including any of the downstream biological effects otherwise resultingfrom the binding to PDGFR of its natural ligand. Such PDGFR kinaseinhibitors include any agent that can block PDGFR activation or any ofthe downstream biological effects of PDGFR activation that are relevantto treating cancer in a patient. Such an inhibitor can act by bindingdirectly to the intracellular domain of the receptor and inhibiting itskinase activity. Alternatively, such an inhibitor can act by occupyingthe ligand binding site or a portion thereof of the PDGF receptor,thereby making the receptor inaccessible to its natural ligand so thatits normal biological activity is prevented or reduced. Alternatively,such an inhibitor can act by modulating the dimerization of PDGFRpolypeptides, or interaction of PDGFR polypeptide with other proteins,or enhance ubiquitination and endocytotic degradation of PDGFR. PDGFRkinase inhibitors include but are not limited to low molecular weightinhibitors, antibodies or antibody fragments, antisense constructs,small inhibitory RNAs (i.e. RNA interference by dsRNA; RNAi), andribozymes. PDGFR kinase inhibitors include anti-PDGF or anti-PDGFRaptamers, anti-PDGF or anti-PDGFR antibodies, or soluble PDGF receptordecoys that prevent binding of a PDGF to its cognate receptor. In apreferred embodiment, the PDGFR kinase inhibitor is a small organicmolecule or an antibody that binds specifically to the human PDGFR. Theability of a compound or agent to serve as a PDGFR kinase inhibitor maybe determined according to the methods known in art and, further, as setforth in, e.g., Dai et al., (2001) Genes & Dev. 15: 1913-25; Zippel, etal., (1989) Eur. J. Cell Biol. 50(2):428-34; and Zwiller, et al., (1991)Oncogene 6: 219-21.

The invention includes PDGFR kinase inhibitors known in the art as wellas those supported below and any and all equivalents that are within thescope of ordinary skill to create. For example, inhibitory antibodiesdirected against PDGF are known in the art, e.g., those described inU.S. Pat. Nos. 5,976,534, 5,833,986, 5,817,310, 5,882,644, 5,662,904,5,620,687, 5,468,468, and PCT WO 2003/025019, the contents of which areincorporated by reference in their entirety. In addition, the inventionincludes N-phenyl-2-pyrimidine-amine derivatives that are PDGFR kinaseinhibitors, such as those disclosed in U.S. Pat. No. 5,521,184, as wellas WO2003/013541, WO2003/078404, WO2003/099771, WO2003/015282, andWO2004/05282 which are hereby incorporated in their entirety byreference.

Small molecules that block the action of PDGF are known in the art,e.g., those described in U.S. Pat. Nos. or Published Application Nos.6,528,526 (PDGFR tyrosine kinase inhibitors), 6,524,347 (PDGFR tyrosinekinase inhibitors), 6,482,834 (PDGFR tyrosine kinase inhibitors),6,472,391 (PDGFR tyrosine kinase inhibitors), 6,949,563, 6,696,434,6,331,555, 6,251,905, 6,245,760, 6,207,667, 5,990,141, 5,700,822,5,618,837, 5,731,326, and 2005/0154014, and International PublishedApplication Nos. WO 2005/021531, WO 2005/021544, and WO 2005/021537, thecontents of which are incorporated by reference in their entirety.

Proteins and polypeptides that block the action of PDGF are known in theart, e.g., those described in U.S. Pat. Nos. 6,350,731 (PDGF peptideanalogs), 5,952,304, the contents of which are incorporated by referencein their entirety.

Bis mono- and bicyclic aryl and heteroaryl compounds which inhibit EGFand/or PDGF receptor tyrosine kinase are known in the art, e.g., thosedescribed in, e.g. U.S. Pat. Nos. 5,476,851, 5,480,883, 5,656,643,5,795,889, and 6,057,320, the contents of which are incorporated byreference in their entirety.

Antisense oligonucleotides for the inhibition of PDGF are known in theart, e.g., those described in U.S. Pat. Nos. 5,869,462, and 5,821,234,the contents of each of which are incorporated by reference in theirentirety.

Aptamers (also known as nucleic acid ligands) for the inhibition of PDGFare known in the art, e.g., those described in, e.g., U.S. Pat. Nos.6,582,918, 6,229,002, 6,207,816, 5,668,264, 5,674,685, and 5,723,594,the contents of each of which are incorporated by reference in theirentirety.

Other compounds for inhibiting PDGF known in the art include thosedescribed in U.S. Pat. Nos. 5,238,950, 5,418,135, 5,674,892, 5,693,610,5,700,822, 5,700,823, 5,728,726, 5,795,910, 5,817,310, 5,872,218,5,932,580, 5,932,602, 5,958,959, 5,990,141, 6,358,954, 6,537,988 and6,673,798, the contents of each of which are incorporated by referencein their entirety.

A number of types of tyrosine kinase inhibitors that are selective fortyrosine kinase receptor enzymes such as PDGFR are known (see, e.g.,Spada and Myers ((1995) Exp. Opin. Ther. Patents, 5: 805) and Bridges((1995) Exp. Opin. Ther. Patents, 5: 1245). Additionally Law and Lydonhave summarized the anticancer potential of tyrosine kinase inhibitors((1996) Emerging Drugs: The Prospect For Improved Medicines, 241-260).For example, U.S. Pat. No. 6,528,526 describes substituted quinoxalinecompounds that selectively inhibit platelet-derived growthfactor-receptor (PDGFR) tyrosine kinase activity. The known inhibitorsof PDGFR tyrosine kinase activity includes quinoline-based inhibitorsreported by Maguire et al., ((1994) J. Med. Chem., 37: 2129), and byDolle, et al., ((1994) J. Med. Chem., 37: 2627). A class ofphenylamino-pyrimidine-based inhibitors was recently reported byTraxler, et al., in EP 564409 and by Zimmerman et al., ((1996) Biorg.Med. Chem. Lett., 6: 1221-1226) and by Buchdunger, et al., ((1995) Proc.Nat. Acad. Sci. (USA), 92: 2558). Quinazoline derivatives that areuseful in inhibiting PDGF receptor tyrosine kinase activity includebismono- and bicyclic aryl compounds and heteroaryl compounds (see,e.g., WO 92/20642), quinoxaline derivatives (see (1994) Cancer Res., 54:6106-6114), pyrimidine derivatives (Japanese Published PatentApplication No. 87834/94) and dimethoxyquinoline derivatives (seeAbstracts of the 116th Annual Meeting of the Pharmaceutical Society ofJapan (Kanazawa), (1996), 2, p. 275, 29(C2) 15-2).

Specific preferred examples of low molecular weight PDGFR kinaseinhibitors that can be used according to the present invention includeImatinib (GLEEVEC®; Novartis); SU-12248 (sunitib malate, SUTENT®;Pfizer); Dasatinib (SPRYCEL®; BMS; also known as BMS-354825); Sorafenib(NEXAVAR®; Bayer; also known as Bay-43-9006); AG-13736 (Axitinib;Pfizer); RPR127963 (Sanofi-Aventis); CP-868596 (Pfizer/OSIPharmaceuticals); MLN-518 (tandutinib; Millennium Pharmaceuticals);AMG-706 (Motesanib; Amgen); ARAVA® (leflunomide; Sanofi-Aventis; alsoknown as SU101), and OSI-930 (OSI Pharmaceuticals); Additional preferredexamples of low molecular weight PDGFR kinase inhibitors that are alsoFGFR kinase inhibitors that can be used according to the presentinvention include XL-999 (Exelixis); SU6668 (Pfizer); CHIR-258/TKI-258(Chiron); RO4383596 (Hoffmann-La Roche) and BIBF-1120 (BoehringerIngelheim).

As used herein, the term “FGFR kinase inhibitor” refers to any FGFRkinase inhibitor that is currently known in the art or that will beidentified in the future, and includes any chemical entity that, uponadministration to a patient, results in inhibition of a biologicalactivity associated with activation of the FGF receptor in the patient,including any of the downstream biological effects otherwise resultingfrom the binding to FGFR of its natural ligand. Such FGFR kinaseinhibitors include any agent that can block FGFR activation or any ofthe downstream biological effects of FGFR activation that are relevantto treating cancer in a patient. Such an inhibitor can act by bindingdirectly to the intracellular domain of the receptor and inhibiting itskinase activity. Alternatively, such an inhibitor can act by occupyingthe ligand binding site or a portion thereof of the FGF receptor,thereby making the receptor inaccessible to its natural ligand so thatits normal biological activity is prevented or reduced. Alternatively,such an inhibitor can act by modulating the dimerization of FGFRpolypeptides, or interaction of FGFR polypeptide with other proteins, orenhance ubiquitination and endocytotic degradation of FGFR. FGFR kinaseinhibitors include but are not limited to low molecular weightinhibitors, antibodies or antibody fragments, antisense constructs,small inhibitory RNAs (i.e. RNA interference by dsRNA; RNAi), andribozymes. FGFR kinase inhibitors include anti-FGF or anti-FGFRaptamers, anti-FGF or anti-FGFR antibodies, or soluble FGFR receptordecoys that prevent binding of a FGFR to its cognate receptor. In apreferred embodiment, the FGFR kinase inhibitor is a small organicmolecule or an antibody that binds specifically to the human FGFR.Anti-FGFR antibodies include FR1-H7 (FGFR-1) and FR3-D11 (FGFR-3)(Imclone Systems, Inc.).

FGFR kinase inhibitors also include compounds that inhibit FGFR signaltransduction by affecting the ability of heparan sulfate proteoglycansto modulate FGFR activity. Heparan sulfate proteoglycans in theextracellular matrix can mediate the actions of FGF, e.g., protectionfrom proteolysis, localization, storage, and internalization of growthfactors (Faham, S. et al. (1998) Curr. Opin. Struct. Biol., 8:578-586),and may serve as low affinity FGF receptors that act to present FGF toits cognate FGFR, and/or to facilitate receptor oligomerization (Galzie,Z. et al. (1997) Biochem. Cell. Biol., 75:669-685).

The invention includes FGFR kinase inhibitors known in the art (e.g.PD173074) as well as those supported below and any and all equivalentsthat are within the scope of ordinary skill to create.

Examples of chemicals that may antagonize FGF action, and can thus beused as FGFR kinase inhibitors in the methods described herein, includesuramin, structural analogs of suramin, pentosan polysulfate,scopolamine, angiostatin, sprouty, estradiol, carboxymethylbenzylaminedextran (CMDB7), suradista, insulin-like growth factor bindingprotein-3, ethanol, heparin (e.g., 6-O-desulfated heparin), lowmolecular weight heparin, protamine sulfate, cyclosporin A, or RNAligands for bFGF.

Other agents or compounds for inhibiting FGFR kinase known in the artinclude those described in U.S. Pat. Nos. 7,151,176 (Bristol-MyersSquibb Company; Pyrrolotriazine compounds); 7,102,002 (Bristol-MyersSquibb Company; pyrrolotriazine compounds); 5,132,408 (Salk Institute;peptide FGF antagonists); and 5,945,422 (Warner-Lambert Company;2-amino-substituted pyrido[2,3-d]pyrimidines); U.S. published Patentapplication Nos. 2005/0256154(4-amino-thieno[3,2-c]pyridine-7-carboxylic acid amide compounds); and2004/0204427 (pyrimidino compounds); and published International PatentApplications WO-2007019884 (Merck Patent GmbH;N-(3-pyrazolyl)-N′-4-(4-pyridinyloxy)phenyl)urea compounds);WO-2007009773 (Novartis AG; pyrazolo[1,5-a]pyrimidin-7-yl aminederivatives); WO-2007014123 (Five Prime Therapeutics, Inc.; FGFR fusionproteins); WO-2006134989 (Kyowa Hakko Kogyo Co., Ltd.; nitrogenousheterocycle compounds); WO-2006112479 (Kyowa Hakko Kogyo Co., Ltd.;azaheterocycles); WO-2006108482 (Merck Patent GmbH;9-(4-ureidophenyl)purine compounds); WO-2006105844 (Merck Patent GmbH;N-(3-pyrazolyl)-N′-4-(4-pyridinyloxy)phenyl)urea compounds);WO-2006094600 (Merck Patent GmbH; tetrahydropyrroloquinolinederivatives); WO-2006050800 (Merck Patent GmbH; N,N′-diarylureaderivatives); WO-2006050779 (Merck Patent GmbH; N,N′-diarylureaderivatives); WO-2006042599 (Merck Patent GmbH; phenylurea derivatives);WO-2005066211 (Five Prime Therapeutics, Inc.; anti-FGFR antibodies);WO-2005054246 (Merck Patent GmbH; heterocyclyl amines); WO-2005028448(Merck Patent GmbH; 2-amino-1-benzyl-substituted benzimidazolederivatives); WO-2005011597 (Irm Llc; substituted heterocyclicderivatives); WO-2004093812 (Irm Llc/Scripps;6-phenyl-7H-pyrrolo[2,3-d]pyrimidine derivatives); WO-2004046152 (F.Hoffmann La Roche A G; pyrimido[4,5-e]oxadiazine derivatives);WO-2004041822 (F. Hoffmann La Roche A G; pyrimido[4,5-d]pyrimidinederivatives); WO-2004018472 (F. Hoffmann La Roche AG;pyrimido[4,5-d]pyrimidine derivatives); WO-2004013145 (Bristol-MyersSquibb Company; pyrrolotriazine derivatives); WO-2004009784(Bristol-Myers Squibb Company; pyrrolo[2,1-f][1,2,4]triazin-6-ylcompounds); WO-2004009601 (Bristol-Myers Squibb Company; azaindolecompounds); WO-2004001059 (Bristol-Myers Squibb Company; heterocyclicderivatives); WO-02102972 (Prochon Biotech Ltd./Morphosys AG; anti-FGFRantibodies); WO-02102973 (Prochon Biotech Ltd.; anti-FGFR antibodies);WO-00212238 (Warner-Lambert Company;2-(pyridin-4-ylamino)-6-dialkoxyphenyl-pyrido[2,3-d]pyrimidin-7-onederivatives); WO-00170977 (Amgen, Inc.; FGFR-L and derivatives);WO-00132653 (Cephalon, Inc.; pyrazolone derivatives); WO-00046380(Chiron Corporation; FGFR-Ig fusion proteins); and WO-00015781 (EliLilly; polypeptides related to the human SPROUTY-1 protein).

Specific preferred examples of low molecular weight FGFR kinaseinhibitors that can be used according to the present invention includeRO-4396686 (Hoffmann-La Roche); CHIR-258 (Chiron; also known asTKI-258); PD 173074 (Pfizer); PD 166866 (Pfizer); ENK-834 and ENK-835(both Enkam Pharmaceuticals A/S); and SU5402 (Pfizer). Additionalpreferred examples of low molecular weight FGFR kinase inhibitors thatare also PDGFR kinase inhibitors that can be used according to thepresent invention include XL-999 (Exelixis); SU6668 (Pfizer);CHIR-258/TKI-258 (Chiron); R04383596 (Hoffmann-La Roche), and BIBF-1120(Boehringer Ingelheim).

The present invention also provides a method of identifying an agentthat inhibits epithelial cells from undergoing an epithelial tomesenchymal transition, comprising contacting a sample of cells of theepithelial cell line CFPAC-1 with a test agent to be screened,contacting the sample with a single protein ligand preparation thatinduces an epithelial-to-mesenchymal transition in CFPAC-1 cells,determining whether the test agent inhibits the epithelial cells in thesample from undergoing an epithelial to mesenchymal transition, bycomparing the level of a biomarker whose level is indicative of the EMTstatus of the sample cells to the level of the same biomarker in anidentical sample of CFPAC-1 cells not contacted with the test agent, andthus determining whether the test agent is an agent that inhibits cellsfrom undergoing an epithelial to mesenchymal transition. Agents thatinhibit epithelial cells from undergoing an epithelial to mesenchymaltransition are useful for the treatment of fibrotic disorders resultingin part from EMT transitions, including but not limited to renalfibrosis, hepatic fibrosis, pulmonary fibrosis, and mesotheliomas. Inone embodiment, the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is selected fromOSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agentthat inhibits cells that have undergone an epithelial to mesenchymaltransition, comprising contacting a sample of cells of the epithelialcell line CFPAC-1 with a single protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, contactingthe sample of cells with a test agent to be screened, determiningwhether the test agent inhibits mesenchymal-like CFPAC-1 cell growth,and thus determining whether it is an agent that inhibits the growth ofcells that have undergone an epithelial to mesenchymal transition.Agents that inhibit mesenchymal-like cells are useful for the treatmentof fibrotic disorders resulting in part from EMT transitions, includingbut not limited to renal fibrosis, hepatic fibrosis, pulmonary fibrosis,and mesotheliomas. In one embodiment, the single protein ligand thatinduces an epithelial-to-mesenchymal transition in CFPAC-1 cells isselected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33;PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4. An alternative embodiment ofthis method comprises, after the step of determining whether the testagent inhibits the growth of cells that have undergone an epithelial tomesenchymal transition, the additional steps of determining whether anagent that inhibits mesenchymal-like CFPAC-1 tumor cell growth, alsoinhibits epithelial CFPAC-1 tumor cell growth, and thus determiningwhether it is an agent that specifically inhibits the growth of cellsthat have undergone an epithelial to mesenchymal transition. In anembodiment of the above methods, an agent that inhibits the growth ofcells that have undergone an epithelial to mesenchymal transition isdetermined to do so by stimulating apoptosis of said cells. In anotherembodiment of the above methods, an agent that inhibits the growth ofcells that have undergone an epithelial to mesenchymal transition isdetermined to do so by inhibiting proliferation of said cells.

The present invention also provides a method of identifying an agentthat stimulates mesenchymal-like cells to undergo a mesenchymal toepithelial transition, comprising contacting a sample of cells of theepithelial cell line CFPAC-1 with a single protein ligand preparation toinduce an epithelial-to-mesenchymal transition in the CFPAC-1 cells,contacting the sample of cells with a test agent to be screened,determining whether the test agent stimulates the mesenchymal-likeCFPAC-1 cells in the sample to undergo a mesenchymal to epithelialtransition, by comparing the level of a biomarker whose level isindicative of the EMT status of the sample cells to the level of thesame biomarker in an identical sample of mesenchymal-like CFPAC-1 cellsnot contacted with the test agent, and thus determining whether the testagent is an agent that stimulates mesenchymal-like cells to undergo amesenchymal to epithelial transition. Agents that stimulatemesenchymal-like cells to undergo a mesenchymal to epithelial transitionare useful for the treatment of fibrotic disorders resulting in partfrom EMT transitions, including but not limited to renal fibrosis,hepatic fibrosis, pulmonary fibrosis, and mesotheliomas. In oneembodiment, the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is selected fromOSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agentthat inhibits cells from undergoing an epithelial to mesenchymaltransition, comprising contacting a sample of cells of the epithelialcell line CFPAC-1, which have been engineered to inducibly express aprotein that stimulates an epithelial to mesenchymal transition inCFPAC-1 cells, with a test agent to be screened, contacting the samplewith a compound that induces the expression of said protein thatstimulates an epithelial to mesenchymal transition in the engineeredCFPAC-1 cells, determining whether the test agent inhibits the cells inthe sample from undergoing an epithelial to mesenchymal transition, bycomparing the level of a biomarker whose level is indicative of the EMTstatus of the sample cells to the level of the same biomarker in anidentical sample of engineered CFPAC-1 cells not contacted with the testagent, and thus determining whether the test agent is an agent thatinhibits cells from undergoing an epithelial to mesenchymal transition.

The present invention also provides a method of identifying an agentthat inhibits cells that have undergone an epithelial to mesenchymaltransition, comprising: contacting a sample of cells of the epithelialcell line CFPAC-1, which have been engineered to inducibly express aprotein that stimulates an epithelial to mesenchymal transition inCFPAC-1 cells, with a compound that induces the expression of saidprotein such that an epithelial-to-mesenchymal transition is induced inthe cells, contacting the sample of cells with a test agent to bescreened, determining whether the test agent inhibits mesenchymal-likeCFPAC-1 cell growth, and thus determining whether it is an agent thatinhibits the growth of cells that have undergone an epithelial tomesenchymal transition. An alternative embodiment of this methodcomprises, after the step of determining whether the test agent inhibitsthe growth of cells that have undergone an epithelial to mesenchymaltransition, the additional steps of determining whether an agent thatinhibits mesenchymal-like CFPAC-1 cell growth, also inhibits epithelialCFPAC-1 cell growth, and thus determining whether it is an agent thatspecifically inhibits the growth of cells that have undergone anepithelial to mesenchymal transition. In an embodiment of the abovemethods, an agent that inhibits the growth of cells that have undergonean epithelial to mesenchymal transition is determined to do so bystimulating apoptosis of said cells. In another embodiment of the abovemethods, an agent that inhibits the growth of cells that have undergonean epithelial to mesenchymal transition is determined to do so byinhibiting proliferation of said cells.

The present invention also provides a method of identifying an agentthat stimulates mesenchymal-like cells to undergo a mesenchymal toepithelial transition, comprising contacting a sample of cells of theepithelial cell line CFPAC-1, which have been engineered to induciblyexpress a protein that stimulates an epithelial to mesenchymaltransition in CFPAC-1 cells, with a compound that induces the expressionof said protein such that an epithelial-to-mesenchymal transition isinduced in the cells, contacting the sample of cells with a test agentto be screened, determining whether the test agent stimulates themesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal toepithelial transition, by comparing the level of a biomarker whose levelis indicative of the EMT status of the sample cells to the level of thesame biomarker in an identical sample of mesenchymal-like CFPAC-1 cellsnot contacted with the test agent, and thus determining whether the testagent is an agent that stimulates mesenchymal-like cells to undergo amesenchymal to epithelial transition.

This invention will be better understood from the Experimental Detailsthat follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter, and are not to be considered in any way limited thereto.

Experimental Details:

Introduction

Models for the identification of targeted anti-cancer agents and therational design of specific combinations of anti-cancer agents areclearly needed to advance such agents to clinical testing. Here wedescribe model systems for both epithelial and mesenchymal tumor cellcomponents. The ability to target both epithelial and mesenchymal celltypes within tumors will be critical to a therapeutic impact on longterm patient survival. The models described enable evaluation ofanti-cancer compounds, antibodies, aptamers and other therapeuticnucleic acids toward the tumors cell types in vitro and in animalmodels.

Materials and Methods

Cell Culture & Treatments

The CFPAC1 cell line was cultured in DMEM media (Gibco #11960)supplemented with 10% FBS (Sigma), 2 mM L Glutamine (Gibco), at 37° C.,5% CO₂. For 7 day treatments, cells were seeded on tissue cultureplastic in complete growth media and allowed to attach overnight at celldensities that would provide approximately 60-80% confluence at end ofexperiment. Ligand and/or inhibitor was added the following day (Day 0)in complete growth media and incubated at 37° C., 5% CO₂. Ligand sourceand concentrations used are as follows: [1] From R&D systems, SDF-1α(stromal derived factor-1 alpha) at 100 ng/ml, TRANCE (TNF-relatedactivation induced cytokine) at 50 ng/ml, FGF1 (fibroblast growth factoracidic) at 10 ng/ml, FGF2 (fibroblast growth factor basic) at 10 ng/ml,BMP4 (bone morphogenetic protein 4) at 100 ng/ml, BMP7 (bonemorphogenetic protein 7) at 100 ng/ml, IGF2 (insulin-like growth factor2) at 100 ng/ml, OSM (Oncostatin M) at 100 ng/ml, NRG1 (Neuregulin-1) at50 ng/ml, PDGFaa (Platelet derived growth factor a) at 50 ng/ml, PDGFbb(platelet derive growth factor b) at 30 ng/ml, Ang2 (Angiopoeitin 2) at400 ng/ml, amphiregulin at 100 ng/ml, HMGB 1 (Human high-mobility groupbox 1 protein) at 50 ng/ml; [2] From Genscript, LIF (Leukemia inhibitoryfactor) at 20 ng/ml, GM-CSF (Granulocyte macrophage colony stimulatingfactor) at 25 ng/ml, M-CSF (Macrophage colony stimulating factor) at 50ng/ml; [3] From Anaspec, PAR1 (Protease activated receptor 1 agonist) at100 μM; PAR2 (Protease activated receptor 2 agonist; peptide SLIGKV-NH₂,Cat. No. 60217-1) at 100 μM; PAR4 (Protease activated receptor δagonist; peptide AYPGKF-NH₂, Cat. No. 60218-1) at 100 μM; [4] FromPeprotech, HGF (Hepatocyte Growth Factor) at 100 ng/ml; IL-31(Interleukin-31) at 50 ng/ml, IL-33 (Interleukin-33) at 50 ng/ml, IL1-a(Interleukin-1 alpha) at 10 ng/ml, CTGF (Connective tissue growthfactor) at 20 ng/ml, WISP1 (Wnt-1 inducible signaling pathway protein)at 20 ng/ml, PTHrP (Polypeptide hormone-related protein) at 50 ng/ml,MCP1 (monocyte chemotactic protein-1) at 50 ng/ml; and [5] from Sigma,Fulvestrant at 1 μM. Fresh medium containing ligand and/or inhibitor wasadded 3-4 days after initial dose, and at Day 7 cells were harvested forwestern blot analysis.

Pharmacological Inhibitors

The following inhibitors were purchased from EMD Biosciences: JAKinhibitor, 1 (catalog #420099) used at 0.25 μM and MEK inhibitor 1(catalog #444937) used at 3 μM.

Western Blotting and Antibodies

Cells were harvested by washing once with PBS, and lysing on ice for 10minutes with scraping (using a Cell Lifter, Corning Inc. #3008) in icecold RIPA buffer (Sigma R0278) containing Protease inhibitor (SigmaP8340), Phosphatase inhibitors (Sigma P2850, P5726) and 200 μM SodiumVanadate. Supernatents were micro-centrifuged at max speed, 4° C. for 10minutes. Protein content was quantitated using the BCA method (Pierce#23225) and samples were normalized to total protein in Laemli bufferand heated at 100° C. for 10 minutes. SDS PAGE was run using a Bis-Trisgel system (Invitrogen NuPAGE) with MOPS buffer (Invitrogen NP0001),transferred onto nitrocellulose using the iBLOT system (Invitrogen) andblocked in PBS-T (0.1% Tween 20), 5% non-fat milk or BSA in guidancewith primary Ab supplier requirements. All primary antibodies wereincubated overnight at 4° C. in PBS-T (0.1% Tween 20) in either 5%non-fat milk or BSA, diluted to the following working dilutions: anti ECadherin (Santa Cruz #sc-21791) 1:500 dilution; anti Vimentin (BDBiosciences #550513) 1:2000 dilution;); anti GAPDH HRP conjugate (SantaCruz sc-25778) 1:1000 dilution; anti β-Actin (Sigma, A5441) 1:10,000dilution. Secondary HRP labeled antibodies used were: anti Rabbit IgGHRP (Amersham NA934) 1:5000 dilution; anti Mouse IgG HRP (Sigma A2304)1:2000 dilution. Membranes were visualized using the Supersignal ELISAFemto Maximum Sensitivity Substrate (Pierce).

Immunofluorescence/Confocal Microscopy

Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences#15710)/PBS (Gibco#14190) for 10 minutes at room temp, washed with PBSand permeabilized with 0.1% Triton X100 (Sigma P1379) in PBS for 10minutes at room temp. Cells were blocked with 3% BSA/PBS for at least 15minutes then incubated with primary antibody diluted in blocking bufferfor a minimum of two hours at room temperature. Anti E-Cadherin was usedat a 1:75 dilution and anti Vimentin, (Millipore #AB5733) was used at a1:2000 dilution. Cells were washed, incubated with fluorescently-labeledsecondary antibodies for one hour, followed by a 5 minute TO-PRO3nuclear counterstain (Invitrogen #T3605) at room temperature, washed andmounted on slides with Pro-Long Gold anti-fade reagent (Invitrogen#P36934). Secondary fluorescent antibodies used were: anti mouse 488 nm(Invitrogen #A11029) 1:600 dilution; anti chicken TRITC (Chemicon#AP194R) 1:50 dilution. Stained cells were captured on a Leica DMRXEmicroscope/SP2 scanner using Leica Confocal Software (LCS).

Chemotaxis/Invasion Assay

Cells were seeded and stimulated as described in the ligand treatmentsection above, over a 7 day period. On the 7^(th) day cells wereserum-starved in the presence of ligand for 24 hours prior to plating inmodified Boyden chambers (Trevigen Cultrex #3458-096-K). Cells wereplated in serum-free medium in the upper chamber at 50,000 cells/well,with 3× concentration of ligand and 10% FBS in the lower chamber as achemo-attractant. 0% FBS was used as a control for comparison. Formigration assays, membranes were left uncoated; for invasion assays,membranes were coated with type IV collagen or basement membrane extract(provided with kit) 24 hours prior to seeding. After 24 (migration) or48 (invasion) hours, cells that had traversed the membrane and attachedto the underside of the membrane were quantified using calcein-AM readon a fluorescent plate reader (Wallac).

MET/EMT Reversion Experiment

Cells were cultured in complete medium with and without ligand for 14days, during which time the media and ligand treatment was replacedevery 3-4 days. 3 Days prior to the first time point (Day 14), cellswere trypsinised, counted and seeded into plastic 35 mm dishes atappropriate seeding densities for the first 3 collection time points. 3Days prior to the last of the 3 collection time points, the procedurewas repeated for the second 3 collection time points, and so on untilall time points were collected. Ligand was removed from all cultures atDay 14. At the time of harvest, cells were harvested as described in thewestern blotting section above. All samples were micro-centrifuged andstored at −80° C. until the end of the experiment, at which time allsamples were processed for western blotting.

2D Immunofluorescence and Confocal Microscopy

Cells were plated on coverslips and stimulated with ligand the followingday (day 0). On day 4, medium and ligands were refreshed. After 7 days,the cells were fixed in 4% paraformaldehyde (EMS#15701)/PBS (Gibco#14190) for 10 minutes at room temperature, and then permeabilized in0.1% Triton X-100 (Sigma #P1379)/PBS for 10 minutes. Cells were blockedin 3% BSA/PBS for one hour and incubated in primary antibody solution(E-cadherin, Santa Cruz #sc21791 and Vimentin, Chemicon #AB5733 inblocking buffer) for two hours at room temperature. Cells were washed inPBS and incubated in secondary antibody (Invitrogen #A11029 and Chemicon#AP194R) for one hour followed by nuclear counterstain TO-PRO3(Invitrogen #T3605). Coverslips were mounted on slides with Pro-LongGold antifade reagent (Invitrogen P36934). Images were captured on aLeica DMRXE microcope with SP2 scanner using Leica Confocal Software.

Doubling Time:

Cells were plated and treated with ligand for 7 days. On day 7, cellswere counted and re-plated on 6 cm cell culture dishes. On days 3, 5, 7,10 and 12, cells were trypsinized and counted to determine the totalnumber of cells in the dish, in duplicate. Doubling time was calculatedusing the multiple time point method on the websitewww.doubling-time.com.

qPCR:

Cells were plated and treated with ligand for one or 7 days. At the timeof harvest, cells were trypsinized and snap frozen for later use. TotalRNA was isolated using RNAqueous-4 PCR Kit (Ambion, AM1914). For qPCR ontumor material, the tumor samples were homogenized using a small tissuehomogenizer (Ultra-Turrax T8 from IKA-Werke) in 175ul RNA lysis buffer.Homogenization was for 1 minute on ice. RNA was isolated using the SVTotal RNA Isolation System (Promega Cat #Z3100) according to themanufactures instructions. Samples were DNase treated using the TurboDNA-Free kit (Ambion, AM1907) and reverse transcribed using SuperscriptIII (Invitrogen, 18080-044) for qPCR analysis. Taqman primers andLocked-Nucleic Acid probes were designed using ProbeFinder software(Universal Probe library, Roche). For mRNA analysis, real time PCR wasperformed using the ABI 7900HT series PCR machine. Thermocycleconditions were as follows: 50° C. for 2 minutes, 95° C. for 10 minutes,95° C. for 15 seconds, 50° C. for 10 seconds, 60° C. for 1 minute. Datawas collected over 45 cycles and then normalized to GAPDH and furthernormalized to the untreated control sample.

3D Matrigel Culture:

80 μl of cold Growth Factor Reduced Matrigel (BD Biosciences #354230)was plated per well into 8 well chamber slides (Labtek II, Nunc #154534)and solidified at 37° C. Cells were trypsinized, counted and suspendedin complete medium containing 2% Matrigel, to give 5000 cells per 300μl. 300 μl was aliquoted into each well and incubated at 37° C., 5% CO₂overnight. Medium was aspirated and replaced with 120 μl per well ofmedium containing 2% Matrigel and ligand treatments. Cells were grownfor 14 days, feeding every 3-4 days. Cultures were imaged by phasecontrast.

3D Immuno-Fluorescence and Confocal Imaging:

3D cultures were fixed in 2% paraformaldehyde (Electron MicroscopySciences #15710)/PBS (Gibco#14190) for 10-20 minutes at roomtemperature, washed with PBS and permeabilized with 0.1% Triton X100(Sigma #P1379) in PBS for 20 minutes. Cells were blocked with 3% BSA/PBSfor 1 hour then incubated with primary antibody in blocking bufferovernight at room temperature. Dilutions are as follows: anti E-Cadherin(Santa Cruz #sc21791), 1:75; anti Vimentin (Millipore #AB5733), 1:2000.Cells were washed in PBS, and incubated with secondary antibodiesovernight at room temperature in the dark (anti-mouse Alexa Fluor 488nm, Invitrogen #A11029, 1:600; anti-chicken Alexa Fluor 568 nm,Invitrogen #A11041, 1:600). Cells were washed 3×20 minutes in PBS, thethird wash containing 5 μM TO-PRO3 (Invitrogen #T3605), and mounted withPro-Long Gold anti-fade reagent (Invitrogen #P36934). Cells were imagedon a Leica DMRXE microscope/SP2 scanner using Leica Confocal Software(LCS).

In Vivo Growth and Immunohistochemistry:

CFPAC 1 cells (1*10e6) were implanted orthotopically into the pancreasof nude mice. The tumors were allowed to establish and the animalssacrificed at 2, 4, and 8 weeks for evaluation of the tumors. Tumorswere fixed in Formalin overnight, paraffin embedded and sectioned.Sections were stained using Dako reagents and protocols. For E-Cadherinstaining, sections were pretreated with Target Retrieval Solution(#S1699), rinsed with TBS/Tween20 and then blocked first with Peroxidaseblocking reagent (#S2001) then with Protein Blocking solution from theVectastain Elite Rabbit Kit (#PK6106). Sections were rinsed andincubated with primary antibody (E-cadherin CST#3195) diluted at 1:50 inDako antibody diluent (#S0809) for one hour at room temperature.Sections were rinsed, incubated in Vectastain kit rabbit secondaryantibody followed by incubation with Vectastain ABC solution for 30minutes. Sections were rinsed and developed with DAB+Chromogen, thenrinsed, counterstained with Gill's Hematoxylin and mounted. Vimentinstaining was done following the same protocol with the Vectastain EliteGoat kit (PK6105) using Chemicon Vimentin #AB5733 chicken polyclonal at1:6400 dilution, and a solution of rabbit anti-goat and anti-chickensecondary antibody.

Results

EMT Ligand Driven Cell Models

The ability of cell lines to undergo epithelial to mesenchymaltransition (EMT) was investigated using different extracellular liganddrivers. A panel of NSCLC, pancreatic, and breast cancer cell lines werescreened by treatment of cells for 7 days in presence of respectiveligands, either singly or in combination. Initial screening determinedthe ability of the ligand to cause morphology change, to down-regulatethe epithelial marker, e-cadherin or to up-regulate a mesenchymal markersuch as vimentin. Potential EMT cell models should up-regulate amesenchymal marker and display morphology change. More robust modelswould show a down-regulation of e-cadherin expression. For those modelswhere protein levels of e-cadherin were not substantiallydown-regulated, it is postulated that e-cadherin is mislocalized.

The CFPAC-1 pancreatic cell line has the propensity to undergo the EMTprocess with a variety of ligands. FIG. 1 summarizes a proportion ofligands tested that have impact on upregulating vimentin with someligand treatments giving marked downregulation of E-cadherin.

The following conclusions were made. HGF and OSM induce the mostcomplete EMT in CFPAC-1 cells. Partial EMT was observed with BMP7, IGF2,LIF, PAR2 agonist SLIGKV-NH₂, IL-33, PAR4 agonist AYPGKF-NH₂, CTGF, andBMP4.

Studies to further investigate the role of HGF and OSM to drive CFPAC-1cells were performed. CFPAC-1 cells were treated with varyingconcentrations of HGF or OSM for 7 days to determine optimal conditionsthat would exhibit protein marker changes for E-cadherin and vimentin(FIG. 2). Using optimal conditions of HGF and OSM effects of EMT weremonitored for morphology change (FIG. 3), for marker changes b confocal(FIG. 4) and for the ability of cells to increase migration and invasionwas investigated (FIG. 5). As shown, cells that were treated with ligandwere better able to migrate and invade.

The epithelial to mesenchymal transition process for CFPAC-1 cells canbe reversed upon withdrawal of the stimulating ligands, HGF, OSM or withthe combination of HGF and OSM (FIG. 6).

The signaling events for the ability of OSM to trigger EMT in CFPAC-1cells was investigated using inhibitors to the JAK and to MEK todetermine the role of the JAK/STAT and MAPK kinase pathway. The JAKinhibitor could block EMT process and the MEK inhibitor was unable toblock the EMT process (FIG. 7). The conclusion was made that theJAK/STAT pathway plays a more critical role for ability of CFPAC-1 cellsto undergo EMT.

Imaging of EMT In Vitro

To characterize EMT in CFPAC 1 cells on the single-cell level,expression of E-cadherin and vimentin in HGF, OSM and HGF+OSM treatedcells compared to untreated cells (FIG. 8) was examined. The untreatedpopulation showed E-cadherin membrane localization at cell-celljunctions with some cells expressing low levels of vimentin. In cellpopulations treated with HGF or OSM alone, most cells grew as isolatedcells, but some small clusters remained. The majority of cells showedstrong vimentin staining with no E-cadherin. In cells that did expressE-cadherin, it was localized in the cytoplasm where it is considerednon-functional. Cells that were treated with HGF+OSM ligand treatmentshowed fewer clusters than the single ligand-treated populations, andfewer E-cadherin positive cells.

EMT Transcriptional Reprogramming Induced by HGF and OSM.

To more fully characterize the transcriptional changes observed in thismodel, mRNA changes in a panel of established EMT genes were quantified(Table 3). We looked for gene expression changes greater than 3 foldafter 1 and 7 days of ligand treatment to distinguish early and latechanges. Other than E-cadherin, there were no changes in expression ofthe epithelial genes that were examined. Integrin β3 was the mostconsistently changed mesenchymal gene. The EMT transcription factorsthat were upregulated in this model were TWIST, ZEB 1 and ZEB2.

TABLE 3 Transcriptional changes to EMT genes in the CFPAC1 model. HGFOSM HGF + OSM 1 Day 7 Day 1 Day 7 Day 1 Day 7 Day CDH1 NC NC NC −3.06 NC−4.31 MMP7 NC NC NC NC NC NC MTA3 NC NC NC NC NC NC ACTN1 NC NC NC NC NCNC CDH2 NC NC NC NC NC NC ITGB3 NC 3.57 31.39 106.2 23.08 86.19 PLAUR NCNC NC NC NC NC SNAI1 NC NC NC NC NC 3.05 SNAI2 NC NC NC NC NC NC SPARCNC −5.41 NC −33 NC NC TWIST1 NC 612 NC 3.3 −3.28 675 VIM NC NC NC NC NC5.23 ZEB1 NC NC NC 3.00 NC 3.80 ZEB2 NC NC 3.52 13.7 3.45 22.41 CFPAC1cells were treated with ligand for 1 or 7 days. Fold changes in mRNAlevels were determined by qPCR. Only changes greater than 3 fold arereported. NC indicates no change.

Phenotypic Impact of EMT in CFPAC1 Cells

To further characterize phenotypic changes associated with EMT in CFPAC1cells, the doubling time of cells after 7 day ligand treatment wasdetermined, since a slower growth rate is a characteristic ofmesenchymal cells (Table 4). HGF treatment did not increase the doublingtime of the cells, but OSM treatment did result in an increase ofapproximately 50%. HGF+OSM ligand treatment also increased doublingtime, but the difference was not distinguishable from OSM alone.

TABLE 4 Impact of EMT on doubling time in CFPAC1 model. Doubling time(hrs) Untreated 36.0 SD 3.46 HGF 41.2 SD 5.94 OSM 54.7 SD 10.25 HGF +OSM 57.7 SD 3.11 Cells were treated for 7 days with HGF, OSM or HGF +OSM. Doubling times were calculated over the following 12 days. OSM andHGF + OSM caused an increase in doubling time, but HGF alone did not..

Impact of EMT on 3D Growth

Cells were grown suspended in a Matrigel matrix to examine the effectsof EMT in a 3D environment where cells are allowed to form contacts withthe extracellular matrix. After 14 days in culture with or withoutHGF+OSM, the cultures were fixed, and the cells immunostained forE-cadherin and vimentin, and with a nuclear stain to distinguish colonyarchitecture (FIG. 9). Untreated colonies consisted of multiple roundnodules attached together, with approximately 40% of the coloniesshowing hollow centers in some of the nodules. The colonies showedmembrane E-cadherin staining with low levels of vimentin in some cells.When cells were grown in the presence of HGF+OSM, the colonyarchitecture became less organized with almost all colonies showingcells growing in the center and increased vimentin staining.

In Vivo Growth

It was determined whether CFPAC I cells underwent EMT in vivo. Untreatedcells were implanted orthotopically and evaluated over time for EMTstatus by western blotting, qPCR and immunohistochemistry. Theorthotopic tumors showed a gradual time-dependent decrease in E-cadherinprotein and mRNA levels from week 2 to week 8 (FIG. 10A). Conversely,the tumors showed an increase in vimentin protein and mRNA from week 2to week 8. Serial sections of the orthotopic tumors demonstrated thatthe tumors contain a mixture of cells with expression of E-cadherin,vimentin, or both (FIG. 10B). Groupings of cells within the tumors(arrows) may express: 1) E-cadherin but not vimentin; 2) Vimentin butnot E-cadherin; or 3) both vimentin and Ecadherin. The loss ofE-cadherin and gain of vimentin is a classical characteristic of EMT.However, during the EMT process cells may express both markers, perhapsrepresenting cells which have undergone partial or incomplete EMT. Thus,CFPAC tumors consist of cells which are potentially in all three stagesof EMT. Over time, more cells in the tumor may progress through the EMTprocess to change the overall status of the tumor as shown by the globaldecrease in E-cadherin and increase in vimentin when using whole tumoranalysis of protein or mRNA. This CFPAC 1 orthotopic animal model may beused in the identification or characterization of new pharmacologicalagents that inhibit tumor cells from undergoing an epithelial tomesenchymal transition, and thus have potential value as anti-canceragents.

ABBREVIATIONS

EGF, epidermal growth factor; EGFR, epidermal growth factor receptor;EGFR-TKI, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor;EMT, epithelial-to-mesenchymal transition; MET,mesenchymal-to-epithelial transition; NSCL, non-small cell lung; NSCLC,non-small cell lung cancer; HNSCC, head and neck squamous cellcarcinoma; CRC, colorectal cancer; MBC, metastatic breast cancer; Brk,Breast tumor kinase (also known as protein tyrosine kinase 6 (PTK6));FCS, fetal calf serum; LC, liquid chromatography; MS, mass spectrometry;IGF-1, insulin-like growth factor-1; TGFα, transforming growth factoralpha; HB-EGF, heparin-binding epidermal growth factor; LPA,lysophosphatidic acid; IC₅₀, half maximal inhibitory concentration; pY,phosphotyrosine; wt, wild-type; PI3K, phosphatidyl inositol-3 kinase;GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activatedprotein kinase; PDK-1, 3-Phosphoinositide-Dependent Protein Kinase 1;Akt, also known as protein kinase B, is the cellular homologue of theviral oncogene v-Akt; mTOR, mammalian target of rapamycin; 4EBP1,eukaryotic translation initiation factor-4E (mRNA cap-binding protein)Binding Protein-1, also known as PHAS-I; p70S6K, 70 kDa ribosomalprotein-S6 kinase; eIF4E, eukaryotic translation initiation factor-4E(mRNA cap-binding protein); Raf, protein kinase product of Raf oncogene;MEK, ERK kinase, also known as mitogen-activated protein kinase kinase;ERK, Extracellular signal-regulated protein kinase, also known asmitogen-activated protein kinase; PTEN, “Phosphatase and Tensinhomologue deleted on chromosome 10”, a phosphatidylinositol phosphatephosphatase; pPROTEIN, phospho-PROTEIN, “PROTEIN” can be any proteinthat can be phosphorylated, e.g. EGFR, ERK, S6 etc; PBS,Phosphate-buffered saline; TGI, tumor growth inhibition; WFI, Water forInjection; SDS, sodium dodecyl sulfate; ErbB2, “v-erb-b2 erythroblasticleukemia viral oncogene homolog 2”, also known as HER-2; ErbB3,“v-erb-b2 erythroblastic leukemia viral oncogene homolog 3”, also knownas HER-3; ErbB4, “v-erb-b2 erythroblastic leukemia viral oncogenehomolog 4”, also known as HER-4; FGFR, Fibroblast Growth FactorReceptor; DMSO, dimethyl sulfoxide.

Incorporation by Reference

All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated herein by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1. A method of identifying an agent that inhibits tumor cells fromundergoing an epithelial to mesenchymal transition, comprisingcontacting a sample of cells of the epithelial tumor cell line CFPAC-1with a test agent to be screened, contacting the sample with a singleprotein ligand preparation that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells, determining whether the test agent inhibitsthe tumor cells in the sample from undergoing an epithelial tomesenchymal transition, by comparing the level of a biomarker whoselevel is indicative of the EMT status of the sample tumor cells to thelevel of the same biomarker in an identical sample of CFPAC-1 cells notcontacted with the test agent, and thus determining whether the testagent is an agent that inhibits tumor cells from undergoing anepithelial to mesenchymal transition.
 2. The method of claim 1, whereinthe single protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from any of the protein ligandsthat bind to and activate the receptors for OSM; HGF; BMP7; IGF2; LIF;PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4.3. The method of claim 2, wherein the single protein ligand that inducesan epithelial-to-mesenchymal transition in CFPAC-1 cells is selectedfrom OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4agonist AYPGKF-NH₂; CTGF; or BMP4.
 4. The method of claim 3, wherein thesingle protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from HGF and OSM.
 5. The methodof claim 1, wherein the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is either aprotein ligand that binds to a receptor that activates the signaltransduction pathways activated by the binding of oncostatin M to itsreceptor or a ligand that binds to a tyrosine kinase receptor andactivates the same signal transduction pathways that are activated bythe binding of HGF to its receptor.
 6. The method of claim 1, whereinthe biomarker whose level is indicative of the EMT status of the sampletumor cells is an epithelial cell biomarker.
 7. The method of claim 6,wherein the epithelial cell biomarker is E-cadherin, CDH1 promoteractivity, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3.
 8. Themethod of claim 1, wherein the biomarker whose level is indicative ofthe EMT status of the sample tumor cells is a mesenchymal cellbiomarker.
 9. The method of claim 8, wherein the mesenchymal cellbiomarker is vimentin, fibronectin, N-cadherin, CDH1 methylation, zeb1,twist, FOXC2 or snail.
 10. A method of identifying an agent thatinhibits tumor cells that have undergone an epithelial to mesenchymaltransition, comprising contacting a sample of cells of the epithelialtumor cell line CFPAC-1 with a single protein ligand preparation toinduce an epithelial-to-mesenchymal transition in the CFPAC-1 cells,contacting the sample of cells with a test agent to be screened,determining whether the test agent inhibits mesenchymal-like CFPAC-1cell growth, and thus determining whether it is an agent that inhibitsthe growth of tumor cells that have undergone an epithelial tomesenchymal transition.
 11. The method of claim 10, wherein the singleprotein ligand that induces an epithelial-to-mesenchymal transition inCFPAC-1 cells is selected from any of the protein ligands that bind toand activate the receptors for OSM; HGF; BMP7; IGF2; LIF; PAR2 agonistSLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4.
 12. Themethod of claim 11, wherein the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is selected fromOSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; and BMP4.
 13. The method of claim 12, wherein thesingle protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from HGF and OSM.
 14. The methodof claim 10, wherein the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is either aprotein ligand that binds to a receptor that activates the signaltransduction pathways activated by the binding of oncostatin M to itsreceptor or a ligand that binds to a tyrosine kinase receptor andactivates the same signal transduction pathways that are activated bythe binding of HGF to its receptor.
 15. The method of claim 10,comprising, after the step of determining whether the test agentinhibits the growth of tumor cells that have undergone an epithelial tomesenchymal transition, the additional steps of determining whether anagent that inhibits mesenchymal-like CFPAC-1 tumor cell growth, alsoinhibits epithelial CFPAC-1 tumor cell growth, and thus determiningwhether it is an agent that specifically inhibits the growth of tumorcells that have undergone an epithelial to mesenchymal transition. 16.The method of claim 10, wherein in the step of determining whether thetest agent inhibits mesenchymal-like CFPAC-1 tumor cell growth, it isdetermined that the test agent does so by stimulating apoptosis of saidtumor cells.
 17. The method of claim 10, wherein in the step ofdetermining whether the test agent inhibits mesenchymal-like CFPAC-1tumor cell growth, it is determined that the test agent does so byinhibiting proliferation of said tumor cells.
 18. A method ofidentifying an agent that stimulates mesenchymal-like tumor cells toundergo a mesenchymal to epithelial transition, comprising contacting asample of cells of the epithelial tumor cell line CFPAC-1 with a singleprotein ligand preparation to induce an epithelial-to-mesenchymaltransition in the CFPAC-1 cells, contacting the sample of cells with atest agent to be screened, determining whether the test agent stimulatesthe mesenchymal-like CFPAC-1 cells in the sample to undergo amesenchymal to epithelial transition, by comparing the level of abiomarker whose level is indicative of the EMT status of the sampletumor cells to the level of the same biomarker in an identical sample ofmesenchymal-like CFPAC-1 cells not contacted with the test agent, andthus determining whether the test agent is an agent that stimulatesmesenchymal-like tumor cells to undergo a mesenchymal to epithelialtransition.
 19. The method of claim 18, wherein the single proteinligand that induces an epithelial-to-mesenchymal transition in CFPAC-1cells is selected from any of the protein ligands that bind to andactivate the receptors for OSM; HGF; BMP7; IGF2; LIF; PAR2 agonistSLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4.
 20. Themethod of claim 19, wherein the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is selected fromOSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonistAYPGKF-NH₂; CTGF; or BMP4.
 21. The method of claim 20, wherein thesingle protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from HGF and OSM.
 22. The methodof claim 18, wherein the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is either aprotein ligand that binds to a receptor that activates the signaltransduction pathways activated by the binding of oncostatin M to itsreceptor or a ligand that binds to a tyrosine kinase receptor andactivates the same signal transduction pathways that are activated bythe binding of HGF to its receptor.
 23. The method of claim 18, whereinthe biomarker whose level is indicative of the EMT status of the sampletumor cells is an epithelial cell biomarker.
 24. The method of claim 23,wherein the epithelial cell biomarker is E-cadherin, CDH1 promoteractivity, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3.
 25. Themethod of claim 18, wherein the biomarker whose level is indicative ofthe EMT status of the sample tumor cells is a mesenchymal cellbiomarker.
 26. The method of claim 25, wherein the mesenchymal cellbiomarker is vimentin, fibronectin, N-cadherin, CDH1 methylation, zeb 1,twist, FOXC2 or snail.
 27. A method of preparing a compositioncomprising a chemical compound which inhibits the growth of tumor cellsthat have undergone an epithelial to mesenchymal transition, whichcomprises contacting a sample of cells of the epithelial tumor cell lineCFPAC-1 with a test agent to be screened, contacting the sample with asingle protein ligand preparation that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells, determiningwhether the test agent inhibits the tumor cells in the sample fromundergoing an epithelial to mesenchymal transition, by comparing thelevel of a biomarker whose level is indicative of the EMT status of thesample tumor cells to the level of the same biomarker in an identicalsample of CFPAC-1 cells not contacted with the test agent, and thusdetermining whether the test agent is an agent that inhibits tumor cellsfrom undergoing an epithelial to mesenchymal transition, and admixingthe test agent so identified with a carrier, thereby preparing saidcomposition.
 28. A method of preparing a composition comprising achemical compound which inhibits the growth of tumor cells that haveundergone an epithelial to mesenchymal transition, which comprisescontacting a sample of cells of the epithelial tumor cell line CFPAC-1with a single or protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, contactingthe sample of cells with a test agent to be screened, determiningwhether the test agent inhibits mesenchymal-like CFPAC-1 cell growth,and thus determining whether it is an agent that inhibits the growth oftumor cells that have undergone an epithelial to mesenchymal transition,and admixing the test agent so identified with a carrier, therebypreparing said composition.
 29. A method of preparing a compositioncomprising a chemical compound which inhibits the growth of tumor cellsthat have undergone an epithelial to mesenchymal transition, whichcomprises contacting a sample of cells of the epithelial tumor cell lineCFPAC-1 with a single or protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, contactingthe sample of cells with a test agent to be screened, determiningwhether the test agent stimulates the mesenchymal-like CFPAC-1 cells inthe sample to undergo a mesenchymal to epithelial transition, bycomparing the level of a biomarker whose level is indicative of the EMTstatus of the sample tumor cells to the level of the same biomarker inan identical sample of mesenchymal-like CFPAC-1 cells not contacted withthe test agent, and thus determining whether the test agent is an agentthat stimulates mesenchymal-like tumor cells to undergo a mesenchymal toepithelial transition, and admixing the test agent so identified with acarrier, thereby preparing said composition.
 30. A mesenchymal-liketumor cell preparation for use in the identification of anti-canceragents, wherein said tumor cell preparation is prepared by a processcomprising: contacting a sample of cells of the epithelial tumor cellline CFPAC-1 with a single protein ligand preparation to induce anepithelial-to-mesenchymal transition in the CFPAC-1 cells, wherein thesingle protein ligand that induces an epithelial-to-mesenchymaltransition in CFPAC-1 cells is selected from any of the protein ligandsthat bind to and activate the receptors for OSM; HGF; BMP7; IGF2; LIF;PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4.31. The cell preparation of claim 30, wherein the single protein ligandthat induces an epithelial-to-mesenchymal transition in CFPAC-1 cells isselected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33;PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4.
 32. The cell preparation ofclaim 31, wherein the single protein ligand that induces anepithelial-to-mesenchymal transition in CFPAC-1 cells is selected fromHGF and OSM.
 33. The cell preparation of claim 30, wherein the singleprotein ligand that induces an epithelial-to-mesenchymal transition inCFPAC-1 cells is either a protein ligand that binds to a receptor thatactivates the signal transduction pathways activated by the binding ofoncostatin M to its receptor or a ligand that binds to a tyrosine kinasereceptor and activates the same signal transduction pathways that areactivated by the binding of HGF to its receptor. 34-74. (canceled)
 75. Amethod of identifying an agent that inhibits tumor cells from undergoingan epithelial to mesenchymal transition, comprising: contacting a sampleof cells of the epithelial tumor cell line CFPAC-1 with a test agent tobe screened, wherein the cells are implanted orthotopically into thepancreas of an immune-deficient animal, determining whether the testagent inhibits the tumor cells in the sample from undergoing anepithelial to mesenchymal transition, by comparing the level of abiomarker whose level is indicative of the EMT status of the sampletumor cells to the level of the same biomarker in an identical sample ofCFPAC-1 cells not contacted with the test agent, and thus determiningwhether the test agent is an agent that inhibits tumor cells fromundergoing an epithelial to mesenchymal transition.
 76. The method ofclaim 75, wherein the immune-deficient animal is a nude mouse.
 77. Themethod of claim 75, wherein the biomarker whose level is indicative ofthe EMT status of the sample tumor cells is an epithelial cellbiomarker.
 78. The method of claim 77, wherein the epithelial cellbiomarker is E-cadherin, CDH1 promoter activity, cytokeratin 8,cytokeratin 18, P-cadherin or erbB3.
 79. The method of claim 75, whereinthe biomarker whose level is indicative of the EMT status of the sampletumor cells is a mesenchymal cell biomarker.
 80. The method of claim 79,wherein the mesenchymal cell biomarker is vimentin, fibronectin,N-cadherin, CDH1 methylation, zeb1, twist, FOXC2 or snail.
 81. Thepresent invention also provides an animal model for use in theidentification of anti-cancer agents, which comprises a sample of cellsof the epithelial tumor cell line CFPAC-1 which have been implantedorthotopically into the pancreas of an immuno-deficient animal.
 82. Themethod of claim 81, wherein the immune-deficient animal is a nude mouse.